Therapeutic targeting of the interleukin 1 receptor-associated kinase 4 (IRAK4) in leukemias characterized by rearrangements in the mixed lineage leukemia gene (MLL-r)

ABSTRACT

Disclosed are methods for treating cancers associated with rearrangements in the mixed lineage leukemia gene (MLL-r), including MLL-r leukemia. The methods typically include administering a therapeutic amount of one or more therapeutic agents that inhibit the biological activity of one or more members of the interleukin-1 signaling pathway such inhibitors of interleukin-1 receptor-associated kinase 4 (IRAK4).

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Application No. 62/327,761, filed on Apr. 26, 2016,the content of which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under RO1 CA150265awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

The field of the invention relates to methods for treating cancers. Inparticular, the field of the invention relates to methods, compounds,and compositions for treating cancers characterized by rearrangements inthe mixed lineage leukemia gene, otherwise referred to as “MLL-rcancers,” including MLL-r leukemias. The methods, compounds, andcompositions disclosed herein relate to the use of therapeutic agentsthat inhibit the biological activity of one or more members of theinterleukin-1 signaling pathway, such as inhibitors of interleukin-1receptor kinase 4 (IRAK4).

Rearrangements or translocations of the mixed lineage leukemia gene(MLL-r) have been shown to be associated with aggressive forms ofleukemia. Cases of acute lymphoblastic leukemia (ALL) and acutemyelogenous leukemia (AML) that are characterized by MLL-r are extremelyaggressive and are predominantly seen in infants and in therapy-relatedleukemias. In contrast to other types of leukemias, the prognosis forMLL-r is dismal and despite advances in new therapies, cure rates haveplateaued over the last several years. Therefore, new therapies areneeded.

Chromosomal rearrangements involving translocations between one copy of11q23 and another chromosome can generate oncogenic fusion proteinsconsisting of an n-terminal portion of MLL and c-terminal portion of thefusion partner. The normal in vivo function of MLL is as the enzymaticsubunit of a COMPASS-like complex that methylates histone H3 on itsfourth lysine. The chimeric protein lacks the c-terminalmethyltransferase, but gains properties of the c-terminal fusionpartner. Since many of the translocation partners are transcriptionalactivators, the aberrant recruitment of the translocation partner tonormal MLL targets, which include oncogenes, drives leukemogenesis.Despite the chromosomal translocation, one wild-type copy of the MLLgene exists but the protein levels expressed from this allele are muchlower than the MLL chimeric protein. Therefore, the present inventorshypothesized that a decrease in wild-type MLL protein observed in MLL-rmay contribute to the development of leukemia.

Here, the inventors have shown that the wild-type MLL protein isdecreased in MLL-r leukemia cells. Further, the inventors have shownthat the interleukin-1 signaling pathway regulates the turnover of MLLprotein. By administering inhibitors of the interleukin-1 signalingpathway to leukemia cells lines, including inhibitors of IRAK4, theinventors have determined that levels of wild-type MLL protein can beincreased and growth of MLL-r leukemia cells can be inhibited.Furthermore, inhibitors of inhibitors of the interleukin-1 signalingpathway, including inhibitors of IRAK4, increased survival in a murineleukemia model. In addition to studying inhibitors of the interleukin-1signaling pathway known in the art, the inventors also synthesized newinhibitors of IRAK4. By administering the new inhibitors of IRAK4leukemia cells, the inventors have determined that levels of wild-typeMLL protein can be increased and growth of MLL-r leukemia cells can beinhibited The inventors' results have implications for MLL-r leukemiasas well as other types of cancers which are shown to be characterized byMLL-r.

SUMMARY

Disclosed are methods, compounds, and compositions for treating cancerscharacterized by rearrangements in the mixed lineage leukemia gene,otherwise referred to as “MLL-r cancers.” The disclosed methods include,but are not limited to, treating MLL-r leukemia. The methods includeadministering a therapeutic amount of one or more therapeutic agentsthat inhibit the biological activity of one or more members of theinterleukin-1 signaling pathway to a subject having a cancercharacterized by MLL-r, including inhibitors of interleukin-1 receptorkinase 4 (IRAK4).

Therapeutic agents administered in the disclosed methods may inhibit thebiological activity of one or more members of the interleukin-1signaling pathway, which may include but are not limited to inhibitorsof the interleukin-1 receptor-associated kinase (IRAK), such asinhibitors of IRAK1, IRAK2, IRAK3, and/or IRAK4 in particular.Therapeutic agents administered in the disclosed methods may inhibit thebiological activity of other members of the interleukin-1 signalingpathway such as interleukin-1 ligand α (IL1α), interleukin 1 ligand β(ILβ), interleukin-1 receptor type 1 (IL1R1), interleukin-1 receptoraccessory protein (IL1RAP), toll interacting protein (TOLLIP), myeloiddifferentiation primary response gene 88 (MYD88), tumor necrosis factorreceptor-associated factor 6 (TRAF6), and any combination thereof.

The therapeutic agents may include, but are not limited to, smallmolecule inhibitors, peptide inhibitors, and/or nucleic acid molecules.The therapeutic agents may inhibit the expression and/or activity of theone or more members of the interleukin-1 signaling pathway.

Also disclosed herein are new compounds which may inhibit one or moremembers of the interleukin-1 signaling pathway, such as IRAK4. Thecompounds may be formulated as pharmaceutical compositions, for examplefor treating cancers characterized by rearrangements in the MLL-r genesuch as MLL-r leukemia.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. UBE2O Interacts with an MLL Internal Region and PromotesWild-Type MLL Protein Degradation (A) MLL-rearranged leukemia cell linesSEM (MLL-AFF1) and MM6 (MLL-AF9) have relatively low levels of wild-typeMLL protein compared to the MLL chimeras. Immunoblotting of MLL N320with the D2M7U monoclonal antibody was performed with total cell lysatesof SEM and MM6 cells. (B) Wild-type MLL and MLL fusion alleles aretranscribed at similar levels in SEM and MM6 cells. Total RNA-seq wasperformed with SEM and MM6 cells. Genome browser tracks of MLL RNA areshown, with reads per million (rpm) indicated on the y axis. The SNPsN-terminal to the breakpoint of the MLL gene at chr11:118,317,907 (C>T)seen in SEM cells and at chr11:118,320,259 (A>C) in MM6 cells are shownbelow each track, with the number of sequencing reads from each alleleindicated. (C and D) Identification of UBE2O as anMLL-Inter-specific-interacting protein. (C) Halo-tagged MLL internalregion (MLL-Inter) and C-terminal region (MLL-CT), both of which aremissing in the MLL chimeras, were transiently transfected into HEK293cells. MudPIT analysis identified the UBE2O as the most abundant proteinspecifically interacting with MLL-Inter. Interaction between UBE2O andMLL-Inter was confirmed by co-immunoprecipitation (D). (E) Ectopicexpression of UBE2O induces MLL degradation. HEK293 cells weretransfected with increasing amounts of Myc-UBE2O expression plasmid; 24hr later, cells were treated with DMSO or MG132 for 12 hr. (F) Depletionof UBE2O specifically stabilizes wild-type MLL, but not the MLL chimeraMLL-AF9. UBE2O was depleted with two independent shRNAs in FLAGMLL-AF9HEK293 cells; 4 days after infection, the MLL N320 and MLL-AF9 levelswere determined with the D2M7U antibody. See also FIG. 8.

FIG. 2. Genome-wide shRNA Screen Identifies the IL-1 Pathway inPromoting MLL Degradation through an MLL-UBE2O Interaction (A) Stablytransfected, randomly integrated Halo-MLL HEK293 cells were stained withHaloTag R110 ligand and sorted by flow cytometry to get thelow-expressing (Halo-MLLDim) Halo-MLL cells. (B) Representative sort forshGFP and TRC lentiviral library-infected Halo-MLLDim cells. Halo-MLLDimcells were infected with lentiviral shRNA libraries or shGFP. Flowcytometry sorting was performed to obtain cells with increased Halo-MLLexpression. Gating for cells with increased HaloTag R110 signal isindicated in pink. (C) Identification of the IL-1 pathway in the shRNAscreen. Components of the IL-1 pathway, including IL1R1, IL1RAP, andTOLLIP, were represented in the 303 enriched genes and are indicatedwith a red star. (D-F) Knockdown of IL-1 pathway components TOLLIP (D),MYD88 (E), AND IL1RAP (F) increases endogenous MLL protein. Knockdown ofIL-1 pathway components and changes in MLL N320 protein levels weredetermined by western blotting. Fold changes of MLL N320 proteinrelative to shGFP are indicated. Tubulin serves as a loading control.(G) IL-1b rapidly induces MLL N320 degradation in 293C6 cells, whichhave ectopically expressed IL-1 receptors IL1R and IL1RAP. 293C6 cellswere stimulated with PBS or 50 ng/mL IL-1b for the indicated time.MG-132 was added prior to IL-1b induction. Fold changes relative to 0min are indicated. (H) IL-1b increases MLL-UBE2O interaction.Halo-MLL-Inter and Myc-UBE2O plasmids were cotransfected into 293C6cells; 24 hr later, these cells were stimulated with IL-1b for 30 min inthe presence of MG-132 before MLL purification with HaloLink resin.Myc-UBE2O was detected with anti-Myc antibody and the inputs wereblotted with anti-HaloTag and anti-Myc antibodies. (I) UBE2O depletiondisrupts IL-1b-induced MLL degradation. After UBE2O depletion for 4days, 293C6 cells were stimulated with 50 ng/mL IL-1b for 90 min. (J)IL-1b stimulates UBE2O-mediated MLL-Inter ubiquitination. His-taggedubiquitin, Myc-UBE2O, and Halo-MLL-Inter plasmids were cotransfectedinto 293C6 as indicated; 24 hr later, these cells were stimulated withIL-1b for 45 min in the presence of MG-132. His-tagged ubiquitinatedproteins were purified with Ni-NTA agarose and blotted with anti-HaloTagantibody. (K) IRAK4 directly phosphorylates UBE2O in vitro. 100 ng IRAK4was incubated with eluates (purified from either vector orFLAG-UBE2O-transfected HEK293 cells) in the presence of g-32P ATP. Thephosphorylated IRAK4 and UBE2O proteins were visualized byautoradiography. See also FIG. 9.

FIG. 3. IRAK Inhibition Stabilizes MLL Protein and Increases Genome-wideMLL Occupancy (A) IRAK4 knockdown leads to increased levels ofendogenous MLL protein in HEK293 cells. Fold changes of MLL N320 proteinrelative to shGFP are indicated. (B) IRAK1/4 inhibitor stabilizesendogenous MLL protein. HEK293 cells were treated with 10 mM IRAK1/4inhibitor for the indicated times. Fold changes of MLL N320 proteinrelative to vehicle (DMSO) are indicated. (C) IRAK1/4 inhibitor has noobvious effect on MLL-AF9 stabilization. FLAG-MLL-AF9 HEK293 cells weretreated with 10 mM IRAK1/4 inhibitor for 2 days and subjected to westernblotting with the FLAG monoclonal antibody. (D and E) IRAK1/4 inhibitordecreases MLL-UBE2O interaction. (D) Halo-MLL-Inter-transfected HEK293cells were treated with 10 mMIRAK1/4 inhibitor for 24 hr, purified withHaloLink resin, and subjected to MudPIT analysis. (E) Confirmation ofthe decreased interaction between MLL-Inter and UBE2O upon IRAK1/4inhibition by coimmunoprecipitation. (F) Genome browser tracks ofMLL-N320 D2M7U ChIP-seq at HOXA and FOXC1 loci after DMSO or IRAK1/4inhibitor treatment for 24 hr. IRAK1/4 inhibitor increases MLL occupancyat HOXA and HOXC loci. (G) Heatmap analysis of MLL occupancy afterIRAK1/4 inhibitor treatment in HEK293 cells. Each row represents a peakof MLL occupancy (n=6,250), with rows ordered by decreasing MLLoccupancy in the inhibitor-treated condition. (H) MLL occupancy issignificantly increased after IRAK1/4 inhibitor treatment. Boxplotsdepict the read coverage for all MLL peaks. The p value was calculatedwith the Wilcoxon signed-rank test. See also FIG. 10.

FIG. 4. Stabilization of MLL through IRAK Inhibition or UBE2O DepletionImpedes Cell Proliferation and Deregulates a Gene Regulatory Network inMLL Leukemia (A) IRAK1/4 inhibitor treatment results in slower growth ofMLL leukemia SEM cells but has no effect on non-MLL leukemia REH cells.Viable cells were seeded at 0.2 million/mL, and they were monitored bytrypan blue exclusion staining and counted using a Vi-CELL XR cellcounter. Data are represented as mean±SD (n=3). (B) 500 nM IRAK4inhibitor treatment blocks SEM cell growth, but not REH cell growth.Data are represented as mean±SD (n=3). (C) Venn diagram of deregulatedgenes in SEM cells by IRAK1/4 and IRAK4 inhibitors. 229 genes weredownregulated and 124 genes were upregulated by both inhibitors. (D)Venn diagram of deregulated genes in SEM and REH cells by bothinhibitors. Little overlap was observed between REH and SEM cells. (E)Hierarchical clustering of 227 genes specifically downregulated in SEMcells, but not REH cells, by both inhibitors. Heatmaps of Zscore-normalized values are displayed. (F) Network enrichment analysisby Metascape (Tripathi et al., 2015) of the 227 genes downregulated onlyin SEM cells. Each cluster is represented by different colors and acircle node represents each enriched term. (G) UBE2O depletion and IRAKinhibition affect a common subset of genes in SEM cells. 121 of 227genes downregulated by IRAK inhibition also are decreased after UBE2Oknockdown. Some examples of common downregulated genes are indicated tothe right. Heatmaps represent Z score-normalized values. See also FIG.11.

FIG. 5. Determinants of the Increased Sensitivity of MLL Leukemia Cellsto IRAK Inhibition (A) IRAK4 inhibitor-specific inhibition of MLLleukemia cell proliferation. Multiple MLL leukemia and non-MLL leukemiaor lymphoma cell lines were cultured with different doses of IRAK4inhibitor for 3 days. Data are represented as mean±SD (n=3). (B) Cellgrowth of MLL-AF9-positive AML MM6 cells is inhibited by IRAKinhibition. Data are represented as mean±SD (n=3). (C) Venn diagramanalysis identifies 59 common downregulated genes by both IRAKinhibitors in MLL-AFF1 SEM and MLL-AF9 MM6 cells. The p value wasdetermined with the hypergeometric test. (D) Hierarchical clustering ofthe 59 common genes downregulated in SEM and MM6cells after IRAKinhibition. Some examples of common downregulated genes are indicated tothe right. (E and F) Depletion of LGALS1 (E) and LMO2 (F) results inreduced growth of MM6 cells. MM6 cells were transduced with shGFPcontrol (GFPi) or two different lentiviral shRNA constructs. Afterselection with puromycin for 4 days, viable cells were seeded at 0.2million/mL and cultured for 3 more days before cell viability counting.Data represent the mean±SD (n=3; **p<0.005, one-way ANOVA). See alsoFIG. 12.

FIG. 6. IRAK Inhibition Displaces MLL Chimera and SEC Occupancy at aSubset of MLL Chimera and SEC Target Genes (A) IRAK inhibitors decreaseMLL-AFF1 and SEC occupancy at the LGALS1, GNA15, and LMO2 genes in SEMcells. Genome browser views of MLL-AFF1 (AFF1-CT) and SEC component AFF4occupancy at the LGALS1, GNA15, and LMO2 genes are shown. Red boxesindicate the promoter-proximal regions with decreased MLL-AFF1 and AFF4occupancy. (B and C) We identified 1,311 promoter regions (±3 kb of thetranscription start site [TSS]) in which MLL-AFF1 occupancy was alteredby IRAK inhibitors. These regions are plotted as heatmaps (B) andmetagene plots, and the Wilcoxon signed-rank test was used to show thatMLL-AFF1 occupancy is significantly decreased after IRAK inhibition (C).(D and E) Heatmap (D), metagene, and statistical analysis (E) of AFF4occupancy at the 1,311 promoter regions are shown. (F) RNA-seq genomebrowser track examples indicate that AFF4 knockdown reduces theexpression of the LGALS1, GNA15, and LMO2 genes in SEM cells. See alsoFIG. 13.

FIG. 7. IRAK Inhibition Substantially Delays Disease Progression andImproves Survival of MLL-AF9 Leukemia Mice (A) Schematic of thedevelopment of secondary murine MLL-AF9 leukemia. After transformationof MLL-AF9, c-Kit+ HSPCs were transplanted into lethally irradiatedC57BL/6 mice with 1 3 106 transformed cells and 2 3 105 support cells.Leukemia cells from primary AML mice were isolated and transplanted intosublethally irradiated C57BL/6 mice. Drug treatments were started at day10 or 19 after transplantation. (B) Kaplan-Meier survival curves ofsecondary transplanted C57BL/6 mice after vehicle and IRAK1/4 or IRAK4inhibitor treatment at day 19 (blast phase). Vehicle or IRAK inhibitorswere administered every other day by intraperitoneal injection for atotal of five treatments. Leukemia was confirmed at the endpoint foreach transplant mouse. The number (n) indicates the number of mice ineach group. The p values were calculated using the log rank test. (C)Kaplan-Meier survival curves of vehicle- and IRAK1/4 or IRAK4inhibitor-treated C57BL/6 mice transplanted with 1 3 104 primary MLL-AF9leukemia cells. 10 days after transplantation, vehicle or IRAKinhibitors were administered every other day by intraperitonealinjection for a total of five treatments. The p values were calculatedusing the log rank test. (D) Wright-Giemsa staining of peripheral bloodsmears from vehicle- and IRAK inhibitor-treated leukemic mice at theendpoint. Arrows indicate partially differentiated MLL-AF9 blast cellsafter IRAK inhibition.

FIG. 8. UBE2O Interacts with an MLL Internal Region and PromotesWild-type MLL Protein Degradation, Related FIG. 1 (A and B) Verificationof the specificity of the MLL D2M7U antibody with ChIP-seq with Mll−/−and Mll+/+ MEF cells. Genome browser views of the Hoxa and Hoxc clustersare shown. ChIP-seq in the Mll wild-type MEFs with a second antibody,Bethyl NT86, is shown. ChIP-seq with Pol II N20 antibody in these MEFcells are also shown at these MLL target genes. (C) Low abundance ofwild-type MLL protein in MLL-translocated leukemia cells. MLL-rearrangedleukemia cell lines RS4; 11 and THP1 have relatively low levels ofwild-type MLL protein compared to the MLL chimera proteins.Immunoblotting with the MLL D2M7U antibody was performed with varioustotal leukemia cell lysates. REH and RL are leukemia cell lines that donot have an MLL translocation and serve as controls. (D) UBE2O does notinteract with the MLL N-terminal region shared with the MLL chimeras orwith some of the most common MLL chimeras. Flag-MLL-NT, Flag-MLL-AF9,Flag-MLL-AFF1, Flag-MLL-ELL and Flag-MLL-ENL were purified withAnti-Flag M2 beads and subjected to MudPIT analysis (Lin et al., 2010).Distributed normalized spectral abundance factors (dNSAF) are shown. Xindicates protein not found. (E) Mapping of MLL-UBE2O interactiondomains identifies a region spanning the MLL breakpoint cluster and thefirst PHD finger as being required for the MLLUBE2O interaction. TheMLL-internal region (MLL-Inter), comprising the breakpoint clusterregion through the FYRN domain (T1), was further truncated to removeadditional PHD fingers (T2 and T3). Halo constructs were transfected inHEK293 cells and purified for MudPIT analysis. (F) MLL-Inter (T1) wasfurther truncated and Halo-MLL truncations were transientlyco-transfected with Myc-tagged UBE2O for immunoprecipitation and westernblotting with the Myc-tag antibody. (G) Knockdown of UBE2O has nosignificant effect on MLL mRNA expression. Data are represented asMean±SD (n=3). n.s, no significant difference with the One-Way AVONAtest.

FIG. 9. Genome-wide shRNA Screen Identifies the IL-1 Pathway inPromoting MLL Degradation through an MLL-UBE2O Interaction, Related to(A and B) Halo-MLL can fully reconstitute MLL/COMPASS in HEK293 cells.Biochemical purification of different COMPASS family members withHaloLink resin from transiently transfected HEK293 cells. Halo-purifiedSETD1A, MLL1, MLL2 (KMT2B) and MLLA (KMT2D) were subjected to SDS-PAGE,silver staining and western blotting. Non-transfected cells (HEK293) andvector only (Halo Vector) transfected cells were used as negativecontrols. Antibodies recognizing the common COMPASS subunit RBBP5 wereused to demonstrate that core COMPASS subunits were present in allCOMPASS purifications. In contrast, Menin was only found in the MLL1 andMLL2 purifications. The composition of MLL/COMPASS is also confirmed byMudPIT analysis (B). (C) Flow chart for the generation of Halo-taggedMLLDim cells. After transient transfection of HEK293 cells with Halo-MLLplasmid, cells were selected with G418 for 3 weeks before staining withHaloTag R110 ligand for FACS sorting for low expressing (Halo-MLLDim).(D) Workflow for pooled lentiviral shRNA screening. Halo-MLLDim cellswere infected with lentiviral shRNA libraries or shGFP and selected for1-2 weeks with puromycin. After HaloTag R110 staining, flow cytometrysorting was performed to obtain cells with increased Halo-MLLexpression. shRNAs from the sorted cells were amplified forhigh-throughput sequencing. (E) Pathway analysis of the enriched 303genes from the shRNA library screen identifies the interleukin 1 (IL-1)and cytokine receptor activity as significantly enriched molecularfunction terms. Immune response and regulation are also enriched inbiological process terms. Pathway analysis was performed with PANTHERand the fold enrichments and p values are shown. (F) Depletion of IL-1pathway components does not affect MLL mRNA levels as determined byRT-qPCR. Data are represented as Mean±SD (n=3). n.s, no significantdifference with the One-Way AVONA test.

FIG. 10. IRAK Inhibition Stabilizes MLL Protein and IncreasesGenome-wide MLL Occupancy, Related to FIG. 3 (A) IRAK1/4 inhibitorstabilizes MLL protein from proteasomal degradation in a dose-dependentmanner. HEK293 cells were first treated with different concentrations ofIRAK1/4 inhibitor for 24 hr followed by treatment with DMSO or theproteasome inhibitor MG132 for 12 hr. (B) IRAK1/4 inhibitor increasesthe stability of endogenous MLL. After DMSO or IRAK1/4 inhibitortreatment for 24 hr, HEK293 cells were treated with the proteinbiosynthesis inhibitor cycloheximide for 5 and 10 hr. (C) IRAK1/4inhibition does not stabilize MLL-AFF1 chimeras. Flag-MLL-AFF1 HEK293cells were treated with the IRAK1/4 inhibitor at 10 mM for 2 days andsubjected to western blotting with anti-FLAG. (D) IRAK1/4 inhibitionincreases MLL occupancy as revealed by ChIP-seq. Track examples ofChIP-seq with anti-MLL N320 (Bethyl NT86) at HOXA, HOXC, and FOXC1 locireveal increased MLL occupancy in the presence of the IRAK1/4 inhibitor.(E and F) Heatmap of MLL occupancy in the presence of DMSO or theIRAK1/4 inhibitor. Occupancy in reads per million (rpm)±3 kb around thecenter of peak is shown (N=6250). Genes are ordered by MLL occupancy inthe inhibitor treatment. The log 2 fold change heatmap indicates ageneral increase in MLL occupancy after IRAK1/4 inhibitor treatment (E).The read counts of all the MLL peaks were plotted and the Wilcoxonsigned-rank test was used to calculate the p value (F).

FIG. 11. Stabilization of MLL through IRAK Inhibition or UBE2O DepletionImpedes Cell Proliferation and Deregulates a Gene Regulatory Network inMLL Leukemia, Related to FIG. 4 (A and B) Venn diagram and gene ontologyanalysis of common deregulated genes in REH and SEM cells after IRAK 1/4inhibitor treatment. 238 genes were downregulated and 186 genes wereupregulated in both REH and SEM cells. (C) Hierarchical clustering ofthe 119 genes specifically upregulated in SEM cells by both the IRAK1/4and IRAK4 inhibitor treatments. (D) Gene ontology analysis ofSEM-specific deregulated genes by both IRAK inhibitors. The enrichedterms are shown with p values and FDR-adjusted q-values. (E and F)Depletion of UBE2O preferentially inhibits the MLL leukemic SEM cellgrowth compared to non-MLL REH cells. REH and SEM cells were infectedwith UBE2O shRNA virus and selected with puromycin for 4 days. Viablecells were seeded and monitored for 4 more days. Data represent theMean±SD (n=3). **p<0.005, One-Way ANOVA. (G and H) Ectopic expression ofan MLL-N-terminal region (1-1250aa) blocks MLL leukemic SEM cellproliferation. SEM cells were infected with lentivirus expressing MLL-NT(1-1250 aa). 2 days after lentivirus infection, the SEM cells wereselected with puromycin for 4 days and monitored for GFP expression (G).The viable cells were seeded at day 0, and counted by trypan blueexclusion staining with a cell viability analyzer (H). Data arerepresented as Mean±SD (n=3). **p<0.005, One-Way ANOVA.

FIG. 12. Determinants of the Increased Sensitivity of MLL Leukemia Cellsto IRAK Inhibition, Related to FIG. 5 (A) Depletion of wild-type MLLreduces the sensitivity of MM6 cells to the IRAK1/4 inhibitor. MM6 cellswere depleted with a shRNA targeting the C terminus of MLL for 3 days.Viable cells were seeded at 0.2 million/ml and cultured for 4 days inthe presence or absence of 5 mMIRAK1/4 inhibitor before cell counting.Data represent the Mean±SD (n=3). (B) Hierarchical clustering of the 28genes that are upregulated by the IRAK1/4 and IRAK4 inhibitor treatmentsin SEM and MM6 cells. (C) MYD88, IRAK1 and IRAK4 have higher expressionin MLL ALL patient cells compared to other ALL leukemia patient cells(TARGET ALL microarray data from Cancer UCSC genome database. Project:COG, POG 9906). P values were calculated with the Wilcoxon signed-ranktest. (D) Irak1 and Irak4 expression profiles in iPS cells, HPSC andMLL-AF9 transformed 1st and 2nd leukemia cells from a previouslypublished study (Liu et al., 2014).

FIG. 13. IRAK Inhibition Displaces MLL Chimera and SEC Occupancy at aSubset of MLL Chimera and SEC Target Genes, Related to FIG. 6. (A)MLL-AFF1 chimera shares the same chromatin binding sites with wild-typeMLL and recruits SEC. Heatmap analysis of MLL N320, AFF1-CT and AFF4occupancy in HEK293 and Flag-MLL-AFF1 HEK293 cells. In MLL-AFF1 HEK293cells, the D2M7U antibody could detect both wild-type MLL and MLL-AFF1.Ectopically expressed MLL-AFF1 in HEK293 cells targets the same sites aswild-type MLL as revealed by increased occupancy of both MLL N320 andAFF1-CT. AFF4, a subunit of SEC, is also recruited to these MLL-AFF1binding sites. All protein coding genes with MLL occupancy in eitherHEK293 or MLL-AFF1 HEK293 cells are included in the heatmap. Each rowrepresents a gene with MLL occupancy around the TSS site (N=12300). (B)Metagene plot of MLL, AFF1-CT and AFF4 occupancies in HEK293 andFlag-MLL-AFF1 HEK293 cells. (C) Genome browser RNA-seq tracks at theAFF4 gene with and without AFF4 knockdown in SEM cells. (D) Decreasedexpression of 127 genes in SEM cells by both IRAK inhibition and AFF4knockdown. Some examples of downregulated genes are indicated on theright.

FIG. 14. IRAK Inhibition Substantially Delays Disease Progression andImproves Survival of MLL-AF9 Leukemia Mice, Related to FIG. 7 (A) IRAKinhibitors suppress growth of primary murine MLL-AF9 cells. Differentdosages of IRAK inhibitors were added to the isolated primary MLL-AF9leukemia cells in the presence of IL3, IL6 and SCF. Two days later, theviable leukemia cells were monitored by trypan blue exclusion. **p<0.005(One-Way ANOVA). (B) IRAK inhibitors decrease the colony formationability of primary murine MLL-AF9 leukemia cells in vitro. 1000 primaryMLL-AF9 leukemia cells were seeded and treated with DMSO or IRAKinhibitors at various dosages for the methylcellulose colony formationassay. **p<0.005 (One-Way ANOVA). (C and D) Spleen and liver weights ofvehicle and IRAK inhibitor-treated groups at sacrifice. (E) White bloodcells counts of vehicle and IRAK inhibitor-treated groups at theendpoint. **p<0.005 (One-Way ANOVA).

FIG. 15. Inhibition of IRAK4 kinase activity by NU's IRAK4 kinaseinhibitors. 10 ng IRAK4, 12.5 uM ATP and 500 ng MBP (substrate) wereused in the IRAK4 kinase assay. 100 uM of each inhibitor were usedindividual in the assay and the reaction product ADP was detected by theADP-GLO assay. Relative activity to DMSO is shown.

FIG. 16. Development of novel compounds (NUCC0200618 and NUCC0200554)that stabilize the wild-type MLL protein and slow leukemic cell growththrough IRAK4 inhibition. A) Western blotting for MLL protein levels incells treated with the indicated compounds designed and synthesized atNorthwestern. Protein stabilization is observed to increase by 6 hoursof treatment. B) Luciferase-based IRAK4 kinase assays (Promega) with theindicated doses of the new compounds. C) Cell viability counts after 3days of treatment with the indicated doses of the new compounds.

FIG. 17. Comparison of novel IRAK4 inhibitors (NUCC numbers) withcommercially available IRAK inhibitors (referred to here as IRAK1/4 andIRAK4) on gene expression in leukemia cells as determined by RNA-seq.SEM cells were maintained in IMDM medium with 10% FBS. NUCC0200618 (5 uMor 10 uM), NUCC0200554(10 uM), IRAK1/4 inhibitor (10 uM) and IRAK4inhibitor (500 nM) were used to treat SEM cells for 3 days. SEM cellswere harvested and used for total RNA-seq. A: PCA plot of each groupwith different treatments. NUCC0200618 (10 uM) and IRAK4 inhibitorsgroup together (medium red oval), while NUCC0200554 (10 uM), NUCC0200618(5 uM) and the IRAK1/4 inhibitor have very similar profiles (large redoval), indicating that the Northwestern inhibitors have the same targetspecificity in cells as the commercially available IRAK inhibitors. DMSOtreated cells group together, and far away from inhibitor treated cells(small red oval). B: Distance plot of each group with the indicatedtreatments. C: Heatmap of upregulated genes in any treatment D: Heatmapof downregulated genes in any treatment compared to the vehicle control(DMSO).

DETAILED DESCRIPTION

The present invention is described herein using several definitions, asset forth below and throughout the application.

Definitions

Unless otherwise specified or indicated by context, the terms “a”, “an”,and “the” mean “one or more.” For example, “a compound” should beinterpreted to mean “one or more compounds.”

As used herein, “about,” “approximately,” “substantially,” and“significantly” will be understood by persons of ordinary skill in theart and will vary to some extent on the context in which they are used.If there are uses of these terms which are not clear to persons ofordinary skill in the art given the context in which they are used,“about” and “approximately” will mean plus or minus <10% of theparticular term and “substantially” and “significantly” will mean plusor minus >10% of the particular term.

As used herein, the terms “include” and “including” have the samemeaning as the terms “comprise” and “comprising” in that these latterterms are “open” transitional terms that do not limit claims only to therecited elements succeeding these transitional terms. The term“consisting of,” while encompassed by the term “comprising,” should beinterpreted as a “closed” transitional term that limits claims only tothe recited elements succeeding this transitional term. The term“consisting essentially of,” while encompassed by the term “comprising,”should be interpreted as a “partially closed” transitional term whichpermits additional elements succeeding this transitional term, but onlyif those additional elements do not materially affect the basic andnovel characteristics of the claim.

As used herein, a “subject” may be interchangeable with “patient” or“individual” and means an animal, which may be a human or non-humananimal, in need of treatment, for example, treatment by includeadministering a therapeutic amount of one or more therapeutic agentsthat inhibit the biological activity of one or more members of theinterleukin-1 signaling pathway.

A “subject in need of treatment” may include a subject having a disease,disorder, or condition that is responsive to therapy with an inhibitorof the biological activity of one or more members of the interleukin-1signaling pathway. For example, a “subject in need of treatment” mayinclude a subject having a cell proliferative disease, disorder, orcondition such as cancer. Cancers may include, but are not limited toadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma and particularly cancers of the adrenal gland, bladder,blood, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia,gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,pancreas, parathyroid, prostate, skin, testis, thymus, and uterus.

A “subject in need of treatment” may include a subject having a cancerthat is characterized by a rearrangement in the mixed lineage leukemiagene, a so-called MLL-r cancer, that is responsive to therapy with aninhibitor of the biological activity of one or more members of theinterleukin-1 signaling pathway. In particular, some leukemias such asacute lymphoblastic leukemia (ALL) or acute myelogenous leukemia (AML)have been shown to be characterized by MLL-r. However, the presentinventors' findings may be applicable to other cancers that arecharacterized by MML-r other than ALL and AML, including, but notlimited to adenocarcinoma, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma which are shown to be characterized by MLL-r. Thepresent inventors' findings may be applicable to cancers of the adrenalgland, bladder, blood, bone, bone marrow, brain, breast, cervix, gallbladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung,muscle, ovary, pancreas, parathyroid, prostate, skin, testis, thymus,and uterus which are shown to be characterized by MLL-r.

As used herein, the phrase “effective amount” shall mean that drugdosage that provides the specific pharmacological response for which thedrug is administered in a significant number of subjects in need of suchtreatment. An effective amount of a drug that is administered to aparticular subject in a particular instance will not always be effectivein treating the conditions/diseases described herein, even though suchdosage is deemed to be a therapeutically effective amount by those ofskill in the art.

The term “alkyl” as contemplated herein includes a straight-chain orbranched alkyl radical in all of its isomeric forms. Similarly, the term“alkoxy” refers to any alkyl radical which is attached via an oxygenatom (i.e., a radical represented as “alkyl-O—*”). As used herein, anasterick “*” is used to designate the point of attachment for anyradical group or substituent group.

Therapeutic Targeting of the Interleukin-1 Pathway in MLL-r Cancers

The present inventors have invented new methods of treating MLL-rcancers, including MLL-r leukemias, in a subject in need thereof. Themethods typically include targeting the interleukin-1 pathway byadministering a therapeutic amount of one or more therapeutic agentsthat inhibit the biological activity of one or more members of theinterleukin-1 signaling pathway.

Therapeutic agents administered in the inventors' may inhibit thebiological activity of one or more members of the interleukin-1signaling pathway, which may include but are not limited tointerleukin-1 receptor-associated kinase 2 (IRAK2), interleukin-1receptor-associated kinase 3 (IRAK3), interleukin-1 receptor-associatedkinase 4 (IRAK4), interleukin-1 ligand α (IL1α), interleukin 1 ligand β(IL1β), interleukin-1 receptor type 1 (IL1R1), interleukin-1 receptoraccessory protein (IL1RAP), toll interacting protein (TOLLIP), myeloiddifferentiation primary response gene 88 (MYD88), interleukin-1receptor-associated kinase 1 (IRAK1), tumor necrosis factorreceptor-associated factor 6 (TRAF6), and any combination thereof. Thetherapeutic agents may include, but are not limited to, small moleculeinhibitors and/or peptide inhibitors. The therapeutic agents may includetherapeutic agents known in the art or described herein, such as the newsmall molecule inhibitors of IRAK4 described herein. The therapeuticagents may be formulated as pharmaceutical compositions for treatingcancers associated with the MLL-r gene, such as MLL-r leukemia.

Inhibitors of members of the IL-1 signaling pathway are known in theart. For example, inhibitors of the IL-1 receptor-associated family ofkinases (i.e., the IRAK-type kinases) are known in the art. So-called“acyl-2-aminobenzimidazole compounds” or“N-(1H-benzimidazol-2-yl)-benzamide compounds” have been shown toinhibit the biological activity of IRAK1 and IRAK4. (See Powers et al.,Biorg & Chem. Lett. 16 (2006) 2842-2845, the content of which isincorporated herein by reference in its entirety). As such, therapeuticagents administered in the methods disclosed herein may includeacyl-2-aminobenzimidazole compounds orN-(1H-benzimidazol-2-yl)-benzamide compounds. In some embodiments, thecompounds have the following formula I:

where n is 0, 1, 2, or 3;

-   R is H, alkyl (e.g., C₁-C₆ alkyl), allyl, cycloalkyl (e.g., C₃-C₆    cycloalkyl), alkoxy (e.g., C₁-C₆ alkoxy), alkylester (e.g., C₁-C₆    alkylester), α-(γ-butyrolactone), (2-tetrahydrofuranyl)methyl,    hydroxyl, amino, alkylamino (e.g., C₁-C₆ alkylamino), dialkylamino    (e.g., C₁-C₆ dialkylamino), and morpholinyl (e.g., N-morpholinyl).-   R² is H, alkyl (e.g., C₁-C₆ alkyl), or allyl;-   optionally the compound is substituted at one or more X positions    with alkyl (e.g., C₁-C₆ alkyl), alkoxy (e.g., C₁-C₆ alkyl), halo    (e.g., Cl or F), nitro, carboxyl, alkylester, or SO₂(CH₂)₂Me;-   and optionally the compound is substituted at one or more Y    positions with alkyl (e.g., C₁-C₆ alkyl), alkoxy (e.g., C₁-C₆    alkyl), halo (e.g., Cl or F), nitro, or cyano.

In further embodiments, the compound having formula I is substituted atone or more X positions with a substituent selected from the groupconsisting of 5-Cl, 5-F, 5-CH₃, 5-OCH₃, 5-CO₂CH₃, 5-SO₂(CH₂)₂Me, 5-NO₂,4-NO₂, 5,6-di-F, 5,6-di-Cl, 4,5-di-F, and 5,6-di-CH₃. In even furtherembodiments, the compound having formula I is substituted at one or moreY positions with a substituent selected from the group consisting of3-NO₂, 3-CN, 3-NO₂-4-F, 3-NO₂-4-Me, 3-Cl, 3,4-di-Cl, 4-NO₂, and 4-OMe.

Specifically, the compound having formula I may include the followingcompound having formula Ia:

In addition, so-called “indolo[2,3-c]quinolone compounds” have beenshown to inhibit the biological activity of IRAK4. (See Tumey et al.,Biorg & Med. Chem. Lett. 24 (2014) 2066-2072, the content of which isincorporated herein by reference in its entirety). As such, therapeuticagents administered in the methods disclosed herein may includeindolo[2,3-c]quinolone compounds. In some embodiments, the compound hasa formula II:

where R is selected from the group consisting of:

R¹ is cyano or nitro; and

R² is H or alkyl (e.g., C₁-C₆ alkyl).

Specifically, the compound having formula II may include the followingcompound having formula IIa:

Other known inhibitors of the IRAK family of kinases include thiazoleamide compounds, imidazo[1,2-α]pyridine compounds and derivativesthereof (see Buckley et al., Bioorg Med Chem Lett. (2008)18(12):3211-3214; Buckely et al., Biorg Med Chem Lett. (2008)18(12):3291-3295); and Buckley et al. Bioorg Med Chem Lett. (2008)18(12):3656-60, the contents of which are incorporated herein byreference in their entireties), oxazole carboxylic acid amide compoundsand derivatives (see International Published Application No. WO2011/043371, the content of which is incorporated herein by reference inits entirety), heteroaryl substituted pyridyl compounds and derivatives(see International Published Application No. WO 2014/074675, the contentof which is incorporated herein by reference in its entirety),2-aminopyrimidine compounds and derivatives (see International PublishedApplication No. WO 2014/058685, the content of which is incorporatedherein by reference in its entirety), indazolyl triazole compounds andderivatives (see International Published Application No. WO 2012/084704,the content of which is incorporated herein by reference in itsentirety), pyrimidine pyrazolyl compounds and derivatives (seeInternational Published Application No. WO 2014/008992),pyridazinone-amides compounds and derivatives (see InternationalPublished Application No. WO 2014/121931, the content of which isincorporated herein by reference in its entirety), macrocyclicpyridazinone compounds and derivatives (see WO 2014/121942, the contentof which is incorporated herein by reference in its entirety), andpyrazolo [1,5a] pyrimidine and thieno [3,2b] pyrimidine compounds andderivatives (see International Published Application No. WO 2012/007375,the content of which is incorporated herein by reference in itsentirety). As such, these compounds also may be administered astherapeutic agents in the presently disclosed methods.

Further to the prior art inhibitors of IRAK4, the present inventors havesynthesized new compounds that may be utilized in pharmaceuticalcompositions and methods for treating subjects in need thereof. Forexample, the disclosed compounds may be utilized in pharmaceuticalcompositions and methods for treating subjects having a disease ordisorder associated with IRAK4 activity in which the disclosed compoundsfunction as inhibitors of IRAK4.

In some embodiments, the disclosed compounds, which optionally areinhibitors of IRAK4, may be imidazo(1,2-a)pyridine derivatives (e.g.,amido-substituted imidazo(1,2-a)pyridine derivatives). In particular,the disclosed compound may have a formula III:

-   wherein R¹, R², R³, and R⁴ are the same or different and each of R¹,    R², R³, and R⁴ is independently —(CH₂)_(m)R′, wherein m is 0-6 and    R′ is selected from hydrogen, halo, amino or alkyl-substituted amino    (e.g., C₁-C₆ alkylamino or C₁-C₆ dialkylamino), hydroxyl, cyano,    alkyl (e.g., C₁-C₆ alkyl which may be straight chain or branched),    allyl, alkoxy (e.g., C₁-C₆ alkoxy which may be straight chain or    branched), saturated or unsaturated cycloalkyl (e.g., C₃-C₇    cycloalkyl or phenyl) which optionally is substituted with alkyl,    halo, hydroxyl, or amino, and saturated or unsaturated    heterocycloalkyl (e.g., piperidinyl, piperizinyl, morpholinyl,    pyridinyl) which optionally is substituted with alkyl, halo,    hydroxyl, or amino;-   wherein R⁵, R⁶, R⁷, R⁸, and R⁹ are the same or different and each of    R⁵, R⁶, R⁷, R⁸, and R⁹ is independently —(CH₂)_(n)R″, wherein n is    0-6 and R″ is selected from hydrogen, halo, amino or    alkyl-substituted amino (e.g., C₁-C₆ alkylamino or C₁-C₆    dialkylamino), hydroxyl, cyano, alkyl (e.g., C₁-C₆ alkyl which may    be straight chain or branched), allyl, alkoxy (e.g., C₁-C₆ alkoxy    which may be straight chain or branched), saturated or unsaturated    cycloalkyl (e.g., C₃-C₇ cycloalkyl or phenyl) which optionally is    substituted with alkyl, halo, hydroxyl, or amino, and saturated or    unsaturated heterocycloalkyl (e.g., piperidinyl, piperizinyl,    morpholinyl, pyridinyl) which optionally is substituted with alkyl,    halo, hydroxyl, or amino; and-   optionally at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ is    not hydrogen, optionally at least one of R¹, R², R³, R⁴ is    alkyl-heteroalkyl, or optionally at least one of R⁵, R⁶, R⁷, R⁸, and    R⁹ is halo.

In some embodiments, the disclosed compounds have a formula IIIa:

-   wherein R² is —(CH₂)_(m)R′, wherein m is 0-6 and R′ is selected from    halo, amino or alkyl-substituted amino (e.g., C₁-C₆ alkylamino or    C₁-C₆ dialkylamino), hydroxyl, cyano, alkyl (e.g., C₁-C₆ alkyl which    may be straight chain or branched), allyl, alkoxy (e.g., C₁-C₆    alkoxy which may be straight chain or branched), saturated or    unsaturated cycloalkyl (e.g., C₃-C₇ cycloalkyl or phenyl) which    optionally is substituted with alkyl, halo, hydroxyl, or amino, and    saturated or unsaturated heterocycloalkyl (e.g., piperidinyl,    piperizinyl, morpholinyl, pyridinyl) which optionally is substituted    with alkyl, halo, hydroxyl, or amino; and-   wherein R⁵, R⁶, R⁷, R⁸, and R⁹ are the same or different and each of    R⁵, R⁶, R⁷, R⁸, and R⁹ is independently —(CH₂)_(n)R″, wherein n is    0-6 and R″ is selected from hydrogen, halo, amino or    alkyl-substituted amino (e.g., C₁-C₆ alkylamino or C₁-C₆    dialkylamino), hydroxyl, cyano, alkyl (e.g., C₁-C₆ alkyl which may    be straight chain or branched), allyl, alkoxy (e.g., C₁-C₆ alkoxy    which may be straight chain or branched), saturated or unsaturated    cycloalkyl (e.g., C₃-C₇ cycloalkyl or phenyl) which optionally is    substituted with alkyl, halo, hydroxyl, or amino, and saturated or    unsaturated heterocycloalkyl (e.g., piperidinyl, piperizinyl,    morpholinyl, pyridinyl) which optionally is substituted with alkyl,    halo, hydroxyl, or amino.

In further embodiments, the disclosed compounds have a formula IIIb:

-   wherein R′ is selected from halo, amino or alkyl-substituted amino    (e.g., C₁-C₆ alkylamino or C₁-C₆ dialkylamino), hydroxyl, cyano,    alkyl (e.g., C₁-C₆ alkyl which may be straight chain or branched),    allyl, alkoxy (e.g., C₁-C₆ alkoxy which may be straight chain or    branched), saturated or unsaturated cycloalkyl (e.g., C₃-C₇    cycloalkyl or phenyl) which optionally is substituted with alkyl,    halo, hydroxyl, or amino, and saturated or unsaturated    heterocycloalkyl (e.g., piperidinyl, piperizinyl, morpholinyl,    pyridinyl) which optionally is substituted with alkyl, halo,    hydroxyl, or amino; and-   wherein R⁵, R⁶, R⁷, R⁸, and R⁹ are the same or different and each of    R⁵, R⁶, R⁷, R⁸, and R⁹ is independently —(CH₂)_(n)R″, wherein n is    0-6 and R″ is selected from hydrogen, halo, amino or    alkyl-substituted amino (e.g., C₁-C₆ alkylamino or C₁-C₆    dialkylamino), hydroxyl, cyano, alkyl (e.g., C₁-C₆ alkyl which may    be straight chain or branched), allyl, alkoxy (e.g., C₁-C₆ alkoxy    which may be straight chain or branched), saturated or unsaturated    cycloalkyl (e.g., C₃-C₇ cycloalkyl or phenyl) which optionally is    substituted with alkyl, halo, hydroxyl, or amino, and saturated or    unsaturated heterocycloalkyl (e.g., piperidinyl, piperizinyl,    morpholinyl, pyridinyl) which optionally is substituted with alkyl,    halo, hydroxyl, or amino.

In particular, the disclosed compounds may have a formula IIIb whereinR′ is heterocycloalkyl, which optionally is substituted with alkyl,halo, hydroxyl, or amino, and/or at least one of R⁵, R⁶, R⁷, R⁸, and R⁹is halo. For example, the disclosed compound may have a formula selectedfrom:

In some embodiments, the newly disclosed compounds, which optionally areinhibitors of IRAK4, may be substituted imidazole compound derivatives.For example, the disclosed compounds may be benzamido-substituted,phenyl-substituted imidazole compound derivatives. In particular, thedisclosed compound may have a formula IV:

-   wherein R¹, R², R³, R⁴, and R⁵ are the same or different and each of    R¹, R², R³, R⁴, and R⁵ is independently —(CH₂)_(m)R′, wherein m is    0-6 and R′ is selected from hydrogen, halo, amino or    alkyl-substituted amino (e.g., C₁-C₆ alkylamino or C₁-C₆    dialkylamino), hydroxyl, cyano, nitro, alkyl (e.g., C₁-C₆ alkyl    which may be straight chain or branched), allyl, alkoxy (e.g., C₁-C₆    alkoxy which may be straight chain or branched), saturated or    unsaturated cycloalkyl (e.g., C₃-C₇ cycloalkyl or phenyl) which    optionally is substituted with alkyl, halo, hydroxyl, or amino, and    saturated or unsaturated heterocycloalkyl (e.g., piperidinyl,    piperizinyl, morpholinyl, pyridinyl) which optionally is substituted    with alkyl, halo, hydroxyl, or amino;-   wherein R⁶, R⁷, R⁸, R⁹, and R¹⁰ are the same or different and each    of R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ is independently —(CH₂)_(n)R″,    wherein n is 0-6 and R″ is selected from hydrogen, halo, amino or    alkyl-substituted amino (e.g., C₁-C₆ alkylamino or C₁-C₆    dialkylamino), hydroxyl, cyano, nitro, alkyl (e.g., C₁-C₆ alkyl    which may be straight chain or branched), allyl, alkoxy (e.g., C₁-C₆    alkoxy which may be straight chain or branched), saturated or    unsaturated cycloalkyl (e.g., C₃-C₇ cycloalkyl or phenyl) which    optionally is substituted with alkyl, halo, hydroxyl, or amino, and    saturated or unsaturated heterocycloalkyl (e.g., piperidinyl,    piperizinyl, morpholinyl, pyridinyl) which optionally is substituted    with alkyl, halo, hydroxyl, or amino; and-   optionally at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and    R¹⁰ is not hydrogen, optionally at least one of R¹, R², R³, R⁴, and    R⁵ is halo or alkyoxy, or optionally at least one of R⁶, R⁷, R⁸, R⁹,    and R¹⁰ is amino, alkyl-substituted amino, dialkyl-substituted    amino, amido, cyano, nitro, hydroxyl, or halo.

In some embodiments, the compounds may have a formula IVa:

In particular, the disclosed compounds may have a formula selected from:

Inhibitors of the interaction between interleukin-1 ligand α (IL1α)and/or interleukin 1 ligand β (IL1β) and their receptor, theinterleukin-1 receptor type 1 (IL1R1), also are known in the art. Theinterleukin-1 receptor antagonist (IL-1RA) is naturally occurringprotein in humans that inhibits the activity of IL1α and IL1β. Arecombinant form of IL-1RA called “Anakinra” is marketed as a drug fortreating inflammation and cartilage damage associated with rheumatoidarthritis under the trademark Kineret® (Sobi, Inc.) Interleukin-1receptor blockade therapy via administering IL-1RA also has beenproposed for treating perinatal brain injury. (See Rosenweig et al.,Frontiers in Pediatrics, October 2014, Volume 2, Article 108, thecontent of which is incorporated herein by reference in its entirety).Anakinra differs from native human IL-1RA in that Anakinra has anN-terminal methionine residue and Anakinra is not glycosylated. As such,therapeutic agents administered in the methods disclosed herein mayinclude interleukin-1 receptor type 1 antagonists such as IL-1RA orrecombinant or modified forms of IL-1RA such as Anakinra.

Inhibitors of myeloid differentiation primary response gene 88 (MYD88)also are known in the art. For example, a peptidomimetic called “ST2825”has been shown to inhibit MYD88 dimerization. (See Loiarro et al., J.Leukocyte Biol. 2007, 82(4): 801-810, the content of which isincorporated herein by reference in its entirety). “ST2825” is otherwiseknown as 4R,7R,8aR)-1′-[2-[4-[[2-(2,4-dichlorophenoxy)acetyl]amino]phenyl]acetyl]-6-oxospiro[3,4,8,8a-tetrahydro-2H-pyrrolo[2,1-b][1,3]thiazine-7,2′-pyrrolidine]-4-carboxamideand by CAS No. 894787-30-5. As such, therapeutic agents administered inthe methods disclosed herein may include ST2825.

Other small molecule inhibitors of MYD88 have been reported. (See Olsonet al., Scientific Reports, Sep. 18, 2015, 5:14246; the content of whichis incorporate herein by reference in its entirety). These other smallmolecule inhibitors of MYD88 include the following compounds:

As such, therapeutic agents administered in the methods disclosed hereinmay include T5910047, T5996207, and T6167923 and derivatives thereof.

Inhibitory peptides of the biological activity of MYD88 also are knownin the art. (See Loiarro et al., 2005. J. Biol. Chem., 2800: 15809-14;the content of which is incorporated herein by reference in itsentirety). In particular, a peptide called “Pepinh-MYD” has been shownto inhibit homodimerization of MYD88. (See id.). Pepinh-MYD contains asequence of the MYD88 TIR homodimerization domain preceded by a proteintransduction sequence. As such, therapeutic agents administered in themethods disclosed herein may include Pepinh-MYD or derivatives of MYD88comprising the homodimerization domain preceded by a proteintransduction sequence. (See id.).

Small molecule inhibitors of the biological activity of tumor necrosisfactor receptor-associated factor 6 (TRAF6) also are known in the art.(See International Published Applications WO 2008/115259 and WO2014/033122, the contents of which are incorporated herein by referencein their entireties). In particular,3-[(2,5-Dimethylphenyl)amino]-1-phenyl-2-propen-1-one (CAS No.433249-94-6), otherwise referred to as “Compound 6877002,” has beenshown to inhibit the biological activity of TRAF6. (See Chatzigeorgiouet al., PNAS, Feb. 18, 2014, vol. 111, no. 7, p. 2686-2691, the contentof which is incorporate herein by reference in its entirety). As such,therapeutic agents administered in the methods disclosed herein mayinclude Compound 6877002.

In addition to the use of small molecules and peptides for inhibitingthe biological activity of the members of the IL-1 signaling pathway,expression of one or more members of the IL-1 signaling pathway also maybe inhibited via RNA interference (RNAi). As such, therapeutic agentsadministered in the methods disclosed herein may include therapeuticagents for administering RNAi therapy as known in the art. (See, e.g.,Davidson et al., Nat. Rev. Genet. 12, 329-340 (May 2011); Wittrup etal., Nat. Rev. Genet. 16, 543-552 (August 2015); Bobbin et al., Ann.Rev. Pharma Toxic. Vol. 56, 103-122 (January 2016); the contents ofwhich are incorporated herein by reference in their entireties). Nucleicacid inhibitors utilized in the disclosed methods may include, but arenot limited to shRNAs and siRNAs that inhibit expression of one or moremembers of the IL-1 signaling pathway.

Formulations and Administration

The formula of the compounds disclosed herein should be interpreted asencompassing all possible stereoisomers, enantiomers, or epimers of thecompounds unless the formulae indicates a specific stereoisomer,enantiomer, or epimer. The formulae of the compounds disclosed hereinshould be interpreted as encompassing salts, esters, amides, or solvatesthereof of the compounds (e.g., pharmaceutically acceptable salts).

The disclosed therapeutic agents may be effective in inhibiting cellproliferation of cancer cells, including mixed lineage leukemia cells.Cell proliferation and inhibition thereof by the presently disclosedtherapeutic agents may be assessed by cell viability methods disclosedin the art including colorimetric assays that utilize dyes such as MTT,XTT, and MTS to assess cell viability. Preferably, the disclosedtherapeutic agents have an IC₅₀ of less than about 10 μM, 5 μM, 1 μM, or0.5 μM in the selected assay.

The therapeutic agents utilized in the methods disclosed herein may beformulated as pharmaceutical compositions that include: (a) atherapeutically effective amount of one or more of the therapeuticagents as disclosed herein; and (b) one or more pharmaceuticallyacceptable carriers, excipients, or diluents. The pharmaceuticalcomposition may include the therapeutic agent in a range of about 0.1 to2000 mg (preferably about 0.5 to 500 mg, and more preferably about 1 to100 mg). The pharmaceutical composition may be administered to providethe therapeutic agent at a daily dose of about 0.1 to 100 mg/kg bodyweight (preferably about 0.5 to 20 mg/kg body weight, more preferablyabout 0.1 to 10 mg/kg body weight). In some embodiments, after thepharmaceutical composition is administered to a subject (e.g., afterabout 1, 2, 3, 4, 5, or 6 hours post-administration), the concentrationof the therapeutic agent at the site of action is about 2 to 10 μM.

The therapeutic agents utilized in the methods disclosed herein may beformulated as a pharmaceutical composition in solid dosage form,although any pharmaceutically acceptable dosage form can be utilized.Exemplary solid dosage forms include, but are not limited to, tablets,capsules, sachets, lozenges, powders, pills, or granules, and the soliddosage form can be, for example, a fast melt dosage form, controlledrelease dosage form, lyophilized dosage form, delayed release dosageform, extended release dosage form, pulsatile release dosage form, mixedimmediate release and controlled release dosage form, or a combinationthereof.

The therapeutic agents utilized in the methods disclosed herein may beformulated as a pharmaceutical composition that includes a carrier. Forexample, the carrier may be selected from the group consisting ofproteins, carbohydrates, sugar, talc, magnesium stearate, cellulose,calcium carbonate, and starch-gelatin paste.

The therapeutic agents utilized in the methods disclosed herein may beformulated as a pharmaceutical composition that includes one or morebinding agents, filling agents, lubricating agents, suspending agents,sweeteners, flavoring agents, preservatives, buffers, wetting agents,disintegrants, and effervescent agents. Filling agents may includelactose monohydrate, lactose anhydrous, and various starches; examplesof binding agents are various celluloses and cross-linkedpolyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101and Avicel® PH102, microcrystalline cellulose, and silicifiedmicrocrystalline cellulose (ProSolv SMCC™). Suitable lubricants,including agents that act on the flowability of the powder to becompressed, may include colloidal silicon dioxide, such as Aerosil®200,talc, stearic acid, magnesium stearate, calcium stearate, and silicagel. Examples of sweeteners may include any natural or artificialsweetener, such as sucrose, xylitol, sodium saccharin, cyclamate,aspartame, and acsulfame. Examples of flavoring agents are Magnasweet®(trademark of MAFCO), bubble gum flavor, and fruit flavors, and thelike. Examples of preservatives may include potassium sorbate,methylparaben, propylparaben, benzoic acid and its salts, other estersof parahydroxybenzoic acid such as butylparaben, alcohols such as ethylor benzyl alcohol, phenolic compounds such as phenol, or quaternarycompounds such as benzalkonium chloride.

Suitable diluents may include pharmaceutically acceptable inert fillers,such as microcrystalline cellulose, lactose, dibasic calcium phosphate,saccharides, and mixtures of any of the foregoing. Examples of diluentsinclude microcrystalline cellulose, such as Avicel® PH101 and Avicel®PH102; lactose such as lactose monohydrate, lactose anhydrous, andPharmatose® DCL21; dibasic calcium phosphate such as Emcompress®;mannitol; starch; sorbitol; sucrose; and glucose.

Suitable disintegrants include lightly crosslinked polyvinylpyrrolidone, corn starch, potato starch, maize starch, and modifiedstarches, croscarmellose sodium, cross-povidone, sodium starchglycolate, and mixtures thereof.

Examples of effervescent agents are effervescent couples such as anorganic acid and a carbonate or bicarbonate. Suitable organic acidsinclude, for example, citric, tartaric, malic, fumaric, adipic,succinic, and alginic acids and anhydrides and acid salts. Suitablecarbonates and bicarbonates include, for example, sodium carbonate,sodium bicarbonate, potassium carbonate, potassium bicarbonate,magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, andarginine carbonate. Alternatively, only the sodium bicarbonate componentof the effervescent couple may be present.

The therapeutic agents utilized in the methods disclosed herein may beformulated as a pharmaceutical composition for delivery via any suitableroute. For example, the pharmaceutical composition may be administeredvia oral, intravenous, intramuscular, subcutaneous, topical, andpulmonary route. Examples of pharmaceutical compositions for oraladministration include capsules, syrups, concentrates, powders andgranules.

The therapeutic agents utilized in the methods disclosed herein may beadministered in conventional dosage forms prepared by combining theactive ingredient with standard pharmaceutical carriers or diluentsaccording to conventional procedures well known in the art. Theseprocedures may involve mixing, granulating and compressing or dissolvingthe ingredients as appropriate to the desired preparation.

Pharmaceutical compositions comprising the therapeutic agents may beadapted for administration by any appropriate route, for example by theoral (including buccal or sublingual), or parenteral (includingsubcutaneous, intramuscular, intravenous or intradermal) route. Suchformulations may be prepared by any method known in the art of pharmacy,for example by bringing into association the active ingredient with thecarrier(s) or excipient(s).

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

Tablets and capsules for oral administration may be in unit dosepresentation form, and may contain conventional excipients such asbinding agents, for example syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; or acceptable wettingagents such as sodium lauryl sulphate. The tablets may be coatedaccording to methods well known in normal pharmaceutical practice. Oralliquid preparations may be in the form of, for example, aqueous or oilysuspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for reconstitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives, such as suspending agents, for example sorbitol,methyl cellulose, glucose syrup, gelatin, hydroxyethyl cellulose,carboxymethyl cellulose, aluminium stearate gel or hydrogenated ediblefats, emulsifying agents, for example lecithin, sorbitan monooleate, oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, oily esters such as glycerine, propylene glycol, orethyl alcohol; preservatives, for example methyl or propylp-hydroxybenzoate or sorbic acid, and, if desired, conventionalflavoring or coloring agents.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

Illustrative Embodiments

The following embodiments are illustrative and are not intended to limitthe scope of the claimed subject matter.

Embodiment 1. A method for treating a cancer characterized by arearrangement in the mixed lineage leukemia gene (MLL-r) in a subject inneed thereof, the method comprising administering a therapeutic amountof a therapeutic agent that inhibits the biological activity of a memberof the interleukin-1 signaling pathway.

Embodiment 2. The method of embodiment 1, wherein the cancer is MLL-rleukemia.

Embodiment 3. The method of embodiment 1, wherein the member of theinterleukin 1 signaling pathway is selected from the group consisting ofinterleukin-1 ligand α (IL1α), interleukin 1 ligand β (IL1 β),interleukin-1 receptor type 1 (IL1R1), interleukin-1 receptor accessoryprotein (IL1RAP), toll interacting protein (TOLLIP), myeloiddifferentiation primary response gene 88 (MYD88), interleukin-1receptor-associated kinase 1 (IRAK1), interleukin-1 receptor-associatedkinase 2 (IRAK2), interleukin-1 receptor-associated kinase 3 (IRAK3),interleukin-1 receptor-associated kinase 4 (IRAK4), and tumor necrosisfactor receptor-associated factor 6 (TRAF6).

Embodiment 4. The method of embodiment 1, wherein the therapeutic agentinhibits the biological activity of interleukin-1 receptor-associatedkinase 1 (IRAK1) and/or the biological activity of interleukin-1receptor-associated kinase 4 (IRAK4).

Embodiment 5. The method of any of the foregoing embodiments, whereinthe therapeutic agent is an N-(1H-benzimidazol-2-yl)-benzamide compound.

Embodiment 6. The method of any of the foregoing embodiments, whereinthe therapeutic agent is a compound having the formula I:

-   n is 0, 1, 2, or 3;-   R is H, alkyl (e.g., C₁-C₆ alkyl), allyl, cycloalkyl (e.g., C₃-C₆    cycloalkyl), alkoxy (e.g., C1-C6 alkoxy), alkylester,    α-(γ-butyrolactone), (2-tetrahydrofuranyl)methyl, hydroxyl, amino,    C₁-C₆ alkyl amino, C₁-C₆ dialkyl amino, and morpholinyl (e.g.,    N-morpholinyl);-   R² is H, alkyl (e.g., C₁-C₆ alkyl), or allyl;-   optionally the compound is substituted at one or more X positions    with alkyl (e.g., C₁-C₆ alkyl), alkoxy (e.g., C₁-C₆ alkyl), halo    (e.g., Cl or F), nitro, carboxyl, alkylester, or SO₂(CH₂)₂Me;-   and optionally the compound is substituted at one or more Y    positions with alkyl (e.g., C₁-C₆ alkyl), alkoxy (e.g., C₁-C₆    alkyl), halo (e.g., Cl or F), nitro, or cyano.

Embodiment 7. The method of embodiment 6, wherein the compound issubstituted at one or more X positions with a substituent selected fromthe group consisting of 5-Cl, 5-F, 5-CH₃, 5-OCH₃, 5-CO₂CH₃,5-SO₂(CH₂)₂Me, 5-NO₂, 4-NO₂, 5,6-di-F, 5,6-di-Cl, 4,5-di-F, and5,6-di-CH₃.

Embodiment 8. The method of embodiment 6 or 7, wherein the compound issubstituted at one or more Y positions with a substituent selected fromthe group consisting of 3-NO₂, 3-CN, 3-NO₂-4-F, 3-NO₂-4-Me, 3-Cl,3,4-di-Cl, 4-NO₂, and 4-OMe.

Embodiment 9. The method of any of the foregoing embodiments wherein thetherapeutic agent is a compound having the formula Ia:

Embodiment 10. The method of any of embodiments 1-4, wherein thetherapeutic agent is an indolo[2,3-c]quinolone compound.

Embodiment 11. The method of any of embodiments 1-4 and 10, wherein thetherapeutic agent is a compound having the formula II:

-   R is selected from the group consisting of:

-   R¹ is cyano or nitro; and-   R² is H or alkyl (e.g., C₁-C₆ alkyl).

Embodiment 12. The method of any of embodiments 1-4, 10, and 11, whereinthe therapeutic agent is a compound having the formula IIa:

Embodiment 13. The method of any of embodiments 1-4, wherein thetherapeutic agent is a compound selected from the group consisting ofimidazo[1,2-a]pyridine compounds and derivatives, oxazole carboxylicacid amide compounds and derivatives, heteroaryl substituted pyridylcompounds and derivatives, 2-aminopyrimidine compounds and derivatives,indazolyl triazole compounds and derivatives, pyrimidine pyrazolylcompounds and derivatives, pyridazinone-amides compounds andderivatives, macrocyclic pyridazinone compounds and derivatives, andpyrazolo [1,5a] pyrimidine compounds and derivatives, and thieno [3,2b]pyrimidine compounds and derivatives.

Embodiment 14. The method of embodiment 1, wherein the therapeutic agentis an interleukin-1 receptor type 1 antagonist.

Embodiment 15. The method of embodiment 14, wherein the therapeuticagent is Anakira.

Embodiment 16. The method of embodiment 1, wherein the therapeutic agentinhibits the biological activity of myeloid differentiation primaryresponse gene 88 (MYD88).

Embodiment 17. The method of embodiment 15, wherein the therapeuticagent is an inhibitory peptide. (See Loiarro et al., 2005. J. Biol.Chem., 2800: 15809-14; the content of which is incorporated herein byreference in its entirety).

Embodiment 18. The method of embodiment 16, wherein the therapeuticagent is selected from a group consisting of T5910047, T5996207, andT6167923.

Embodiment 19. The method of embodiment 16, wherein the therapeuticagent is ST2825.

Embodiment 20. The method of embodiment 1, wherein the therapeutic agentinhibits the biological activity of tumor necrosis factorreceptor-associated factor 6 (TRAF6).

Embodiment 21. The method of embodiment 20, wherein the therapeuticagent is an inhibitory peptide.

Embodiment 22. The method of embodiment 20, wherein the therapeuticagent is Compound 6877002.

Embodiment 23. The method of any of embodiments 1-4, wherein thetherapeutic agent is an imidazo(1,2-a)pyridine derivative compound(e.g., an amido-substituted imidazo(1,2-a)pyridine derivative compound).

Embodiment 24. The method of embodiments 23, wherein the compound has aformula III:

-   wherein R¹, R², R³, and R⁴ are the same or different and each of R¹,    R², R³, and R⁴ is independently —(CH₂)_(m)R′, wherein m is 0-6 and    R′ is selected from hydrogen, halo, amino or alkyl-substituted amino    (e.g., C₁-C₆ alkylamino or C₁-C₆ dialkylamino), hydroxyl, cyano,    alkyl (e.g., C₁-C₆ alkyl which may be straight chain or branched),    allyl, alkoxy (e.g., C₁-C₆ alkoxy which may be straight chain or    branched), saturated or unsaturated cycloalkyl (e.g., C₃-C₇    cycloalkyl or phenyl) which optionally is substituted with alkyl,    halo, hydroxyl, or amino, and saturated or unsaturated    heterocycloalkyl (e.g., piperidinyl, piperizinyl, morpholinyl,    pyridinyl) which optionally is substituted with alkyl, halo,    hydroxyl, or amino;-   wherein R⁵, R⁶, R⁷, R⁸, and R⁹ are the same or different and each of    R⁵, R⁶, R⁷, R⁸, and R⁹ is independently —(CH₂)_(n)R″, wherein n is    0-6 and R″ is selected from hydrogen, halo, amino or    alkyl-substituted amino (e.g., C₁-C₆ alkylamino or C₁-C₆    dialkylamino), hydroxyl, cyano, alkyl (e.g., C₁-C₆ alkyl which may    be straight chain or branched), allyl, alkoxy (e.g., C₁-C₆ alkoxy    which may be straight chain or branched), saturated or unsaturated    cycloalkyl (e.g., C₃-C₇ cycloalkyl or phenyl) which optionally is    substituted with alkyl, halo, hydroxyl, or amino, and saturated or    unsaturated heterocycloalkyl (e.g., piperidinyl, piperizinyl,    morpholinyl, pyridinyl) which optionally is substituted with alkyl,    halo, hydroxyl, or amino; and-   optionally at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ is    not hydrogen, optionally at least one of R¹, R², R³, R⁴ is    alkyl-heteroalkyl, or optionally at least one of R⁵, R⁶, R⁷, R⁸, and    R⁹ is halo.

Embodiment 25. The method of embodiment 23 or 24, wherein the compoundhas a formula IIIa:

Embodiment 26. The method of any of embodiments 23-25, wherein thecompounds has a formula IIIb:

Embodiments 27. The method of any of embodiments 23-26 wherein thecompound has a formula selected from:

Embodiment 28. The method of any of embodiments 1-4, wherein thecompound is a substituted imidazole compound derivative (e.g.,benzamido, phenyl-substituted imidazole compound derivative).

Embodiment 29. The method of embodiment 28, wherein the compound has aformula IV:

-   wherein R¹, R², R³, R⁴, and R⁵ are the same or different and each of    R¹, R², R³, R⁴, and R⁵ is independently —(CH₂)_(m)R′, wherein m is    0-6 and R′ is selected from hydrogen, halo, amino or    alkyl-substituted amino (e.g., C₁-C₆ alkylamino or C₁-C₆    dialkylamino), hydroxyl, cyano, nitro, alkyl (e.g., C₁-C₆ alkyl    which may be straight chain or branched), allyl, alkoxy (e.g., C₁-C₆    alkoxy which may be straight chain or branched), saturated or    unsaturated cycloalkyl (e.g., C₃-C₇ cycloalkyl or phenyl) which    optionally is substituted with alkyl, halo, hydroxyl, or amino, and    saturated or unsaturated heterocycloalkyl (e.g., piperidinyl,    piperizinyl, morpholinyl, pyridinyl) which optionally is substituted    with alkyl, halo, hydroxyl, or amino;-   wherein R⁶, R⁷, R⁸, R⁹, and R¹⁰ are the same or different and each    of R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ is independently —(CH₂)_(n)R″,    wherein n is 0-6 and R″ is selected from hydrogen, halo, amino or    alkyl-substituted amino (e.g., C₁-C₆ alkylamino or C₁-C₆    dialkylamino), hydroxyl, cyano, nitro, alkyl (e.g., C₁-C₆ alkyl    which may be straight chain or branched), allyl, alkoxy (e.g., C₁-C₆    alkoxy which may be straight chain or branched), saturated or    unsaturated cycloalkyl (e.g., C₃-C₇ cycloalkyl or phenyl) which    optionally is substituted with alkyl, halo, hydroxyl, or amino, and    saturated or unsaturated heterocycloalkyl (e.g., piperidinyl,    piperizinyl, morpholinyl, pyridinyl) which optionally is substituted    with alkyl, halo, hydroxyl, or amino; and-   optionally at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and    R¹⁰ is not hydrogen, optionally at least one of R¹, R², R³, R⁴, and    R⁵ is halo or alkyoxy, or optionally at least one of R⁶, R⁷, R⁸, R⁹,    and R¹⁰ is amino, alkyl-substituted amino, dialkyl-substituted    amino, amido, cyano, nitro, hydroxyl, or halo.

Embodiment 29. The method of embodiment 27 or 28, wherein the compoundhas a formula IVa:

Embodiment 30. The method of any of embodiments 27-29 wherein thecompound has a formula selected from:

Embodiment 31. A compound having a formula III:

-   wherein R¹, R², R³, and R⁴ are the same or different and each of R¹,    R², R³, and R⁴ is independently —(CH₂)_(m)R′, wherein m is 0-6 and    R′ is selected from hydrogen, halo, amino or alkyl-substituted    amino, hydroxyl, cyano, alkyl which may be straight chain or    branched, allyl, alkoxy which may be straight chain or branched,    saturated or unsaturated cycloalkyl which optionally is substituted    with alkyl, halo, hydroxyl, or amino, and saturated or unsaturated    heterocycloalkyl which optionally is substituted with alkyl, halo,    hydroxyl, or amino;-   wherein R⁵, R⁶, R⁷, R⁸, and R⁹ are the same or different and each of    R⁵, R⁶, R⁷, R⁸, and R⁹ is independently —(CH₂)_(n)R″, wherein m is    0-6 and R″ is selected from hydrogen, halo, amino or    alkyl-substituted amino, hydroxyl, cyano, alkyl which may be    straight chain or branched, allyl, alkoxy which may be straight    chain or branched, saturated or unsaturated cycloalkyl which    optionally is substituted with alkyl, halo, hydroxyl, or amino, and    saturated or unsaturated heterocycloalkyl which optionally is    substituted with alkyl, halo, hydroxyl, or amino; and-   optionally at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, and R⁹ is    not hydrogen, optionally at least one of R¹, R², R³, R⁴ is    alkyl-heteroalkyl, or optionally at least one of R⁵, R⁶, R⁷, R⁸, and    R⁹ is halo.

Embodiment 32. The compound of embodiment 31, wherein the compound has aformula IIIa:

Embodiment 33. The compound of embodiment 31 or 32, wherein thecompounds has a formula IIIb:

Embodiment 34. The compound of any of embodiments 31-33, wherein thecompound has a formula selected from:

Embodiment 35. A compound having a formula IV:

-   wherein R¹, R², R³, R⁴, and R⁵ are the same or different and each of    R¹, R², R³, R⁴, and R⁵ is independently —(CH₂)_(m)R′, wherein m is    0-6 and R′ is selected from hydrogen, halo, amino or    alkyl-substituted amino, hydroxyl, cyano, nitro, alkyl which may be    straight chain or branched, allyl, alkoxy which may be straight    chain or branched, saturated or unsaturated cycloalkyl which    optionally is substituted with alkyl, halo, hydroxyl, or amino, and    saturated or unsaturated heterocycloalkyl which optionally is    substituted with alkyl, halo, hydroxyl, or amino;-   wherein R⁶, R⁷, R⁸, R⁹, and R¹⁰ are the same or different and each    of R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ is independently —(CH₂)_(n)R″,    wherein n is 0-6 and R″ is selected from hydrogen, halo, amino or    alkyl-substituted amino, hydroxyl, cyano, nitro, alkyl which may be    straight chain or branched, allyl, alkoxy which may be straight    chain or branched, saturated or unsaturated cycloalkyl which    optionally is substituted with alkyl, halo, hydroxyl, or amino, and    saturated or unsaturated heterocycloalkyl which optionally is    substituted with alkyl, halo, hydroxyl, or amino; and-   optionally at least one of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and    R¹⁰ is not hydrogen, optionally at least one of R¹, R², R³, R⁴, and    R⁵ is halo or alkyoxy, or optionally at least one of R⁶, R⁷, R⁸, R⁹,    and R¹⁰ is amino, alkyl-substituted amino, dialkyl-substituted    amino, amido, cyano, nitro, hydroxyl, or halo.

Embodiment 36. The compound of embodiment 35, wherein the compound has aformula IVa:

Embodiment 37. The compound of embodiment 35 or 36, wherein the compoundhas a formula selected from:

Embodiment 38. A pharmaceutical composition comprising the compound ofany of claims 31-37 and a pharmaceutically acceptable carrier.

EXAMPLES

The following Examples are illustrative and are not intended to limitthe scope of the claimed subject matter.

Example 1

Reference is made to the manuscript Liang et al., “Therapeutic Targetingof MLL Degradation Pathways in MLL-Rearranged Leukemia” Cell, Vol. 168,Issues 1-2, p 59-72, 12 Jan. 2017, the content of which is incorporatedherein by reference in its entirety.

Summary

Chromosomal translocations of the mixed-lineage leukemia (MLL) gene withvarious partner genes result in aggressive leukemia with dismaloutcomes. Despite similar expression at the mRNA level from thewild-type and chimeric MLL alleles, the chimeric protein is more stable.We report that UBE2O functions in regulating the stability of wild-typeMLL in response to interleukin-1 signaling. Targeting wildtype MLLdegradation impedes MLL leukemia cell proliferation, and itdownregulates a specific group of target genes of the MLL chimeras andtheir oncogenic cofactor, the super elongation complex.Pharmacologically inhibiting this pathway substantially delaysprogression, and it improves survival of murine leukemia throughstabilizing wild-type MLL protein, which displaces the MLL chimera fromsome of its target genes and, therefore, relieves the cellular oncogenicaddiction to MLL chimeras. Stabilization of MLL provides us with aparadigm in the development of therapies for aggressive MLL leukemia andperhaps for other cancers caused by translocations.

Introduction

Mixed-lineage leukemias (MLLs), including acute myeloid leukemia (AML)and acute lymphoblastic leukemia (ALL), are very aggressive bloodcancers with unique clinical and biological characteristics, and theyare often lethal due to the development of resistance and high relapserates with established therapies, including hematopoietic stem celltransplantation (Pigneux et al., 2015; Tomizawa et al., 2007). MLLleukemia is character characterized and driven by the recurrenttranslocations of an allele of the MLL gene (MLL, KMT2A) with a varietyof other chromosomes (Mohan et al., 2010b). These MLL rearrangementspredominantly occur in pediatric patients, including 69%-79% of infantleukemia, and they are associated with the poorest prognosis of anyacute leukemia subset (Meyer et al., 2013; Mohan et al., 2010b). MLLleukemia remains a clinical challenge with an urgent need for thedevelopment of more effective targeted therapies for these aggressivecancers.

The MLL gene encodes a member of the complex of proteins associated withSetl (COMPASS) family of enzymes that are conserved from yeast to humanand catalyze methylation of histone H3 lysine 4 (Miller et al., 2001;Shilatifard, 2012; Wang et al., 2009). MLL is a large protein comprisedof about 4,000 amino acids (aa), but, due to proteolytic processing byTaspase I, it primarily exists in cells as two fragments, an N-terminal320-kDa fragment (N320) and a C-terminal 180-kDa (C180) fragment(Yokoyama et al., 2011). MLL plays an essential and non-redundant rolein development and hematopoiesis (Jones et al., 2012; Shilatifard,2012). Homozygous Mll knockout mice are embryonic lethal, while Mllheterozygous mice are haploinsufficient with segmentation defects andhematological abnormalities (Yu et al., 1995).

The MLL gene has been characterized in translocations with over 70different translocation partner genes that generate diverse oncogenicMLL chimeras possessing an MLL N-terminal fragment fused in-frame to aC-terminal portion of the partner (Meyer et al., 2013). Some of the mostcommon fusion partners of MLL in leukemia are subunits of the superelongation complex (SEC) (Lin et al., 2010) and/or the DOT1L complex(DotCom) (Mohan et al., 2010a; Nguyen et al., 2011). The inclusion ofMLL's chromatin-targeting domains in the MLL chimeras, together withtheir fusion to SEC and DotCom subunits, could result in aberrantrecruitment of SEC and/or DotCom to deregulate MLL target genes, leadingto increased proliferation and defects in differentiation (Mohan et al.,2010b; Shilatifard, 2012; Smith et al., 2011).

Gene disruption of the remaining wild-type copy of MLL, but not loss ofhistone methyltransferase activity, has been reported to jeopardize theinitiation of MLL-AF9 leukemia in hematopoietic stem and progenitorcells (HSPCs) (Mishra et al., 2014; Thiel et al., 2010), consistent withthe general requirement of MLL for embryonic hematopoiesis and thespecification of postnatal HSPCs (Gan et al., 2010; Jude et al., 2007;McMahon et al., 2007). However, the existence of patient-derived MLLleukemia cell lines (such as ML2) with a deletion of the entirewild-type MLL locus suggests that wild-type MLL may not be required forthe maintenance of MLL leukemia. Interestingly, in AML patients withpartial tandem duplications of the MLL gene (MLL-PTD), wild-type MLLtranscription is generally silenced and contributes to MLL-PTD-mediatedleukemia (Whitman et al., 2005). However, the interplay betweenwild-type MLL and the MLL chimeras in MLL-rearranged leukemia remainselusive, given the fact that chromatin-interacting domains from the Nterminus are shared between oncogenic MLL chimeras and wild-type MLL.

Here we found that wild-type MLL protein is much less abundant than theMLL chimeras in MLL leukemia cells. Therefore, we reasoned that thestabilization of the wild-type copy of the MLL protein could displaceMLL chimeras from chromatin and, therefore, evade the oncogenicaddiction of these cells to MLL chimeras. To test this hypothesis, weestablished biochemical approaches and a genome-wide small hairpin RNA(shRNA) screen to identify factors regulating MLL protein degradation.These studies identified UBE2O and the interleukin-1 (IL-1) pathway inregulating MLL stability. Disruption of the balance between wild-typeMLL and MLL chimeras through chemical inhibition of the interleukin-1receptor-associated kinases (IRAKs) impedes MLL leukemia cellproliferation both in vitro and in vivo. Together, our study revealsthat targeting MLL/COMPASS degradation pathways is a promising strategyfor treating the aggressive and otherwise refractory MLL leukemia, whilealso providing a new paradigm in the development of therapies throughprotein stabilization that perhaps can be applied for the treatment ofother cancers resulting from translocations.

Results

UBE2O Interacts with an Internal Region of MLL and Promotes Wild-TypeMLL Degradation. We first validated the specificity of the MLLantibodies using wild-type and MLL-null mouse embryonic fibroblast (MEF)cells (FIGS. 8A and 8B; data not shown). To determine the stability ofthe MLL chimera and the wild-type MLL, we analyzed MLL levels inmultiple leukemia cell lines, and we found that the wildtype MLL proteinis much less abundant than the corresponding MLL chimera when assayed bywestern blotting (FIGS. 1A and 8C). To determine if this observation wasa consequence of lower MLL mRNA levels, we used total RNA sequencing(RNAseq) with SEM (MLL-AFF1, also called MLL-AF4) and MM6 (MLL-AF9)leukemia cells to identify MLL allele-specific SNPs that are in regionsN-terminal to the breakpoints. Each SNP had similar levels of RNAexpression (FIG. 1B), suggesting that wild-type MLL protein could beless stable than the MLL chimeras and that stabilization of MLL bytargeting MLL degradation factors might be a potential strategy for MLLleukemia treatment.

MLL chimeras shared the same N terminus with wild-type MLL but lostinternal regions C-terminal of the breakpoint (FIG. 1C). To identifyproteins that associate with these missing regions, we expressed andpurified the Halo-tagged portions of an internal region (MLL-Inter) anda C-terminal region (MLL-CT) in HEK293 cells. Multidimensional proteinidentification technology (MudPIT) analysis of the co-eluted proteinsidentified the ubiquitin-conjugating enzyme E2O (UBE20, an E2/E3ubiquitin ligase) to be the most abundant protein specificallyinteracting with MLL-Inter, which was confirmed byco-immunoprecipitation (FIGS. 1C and 1D). In agreement, UBE2O did notinteract with the most common MLL chimeras (MLL-AF9, MLLAFF1, MLL-ENL,and MLL-ELL) (FIG. 8D) (Lin et al., 2010). Further truncation ofMLL-Inter demonstrated that the region spanning the MLL breakpointregion and the first PHD finger domain (1,163-1,482 aa) was required andsufficient for the MLL-UBE2O interaction (FIGS. 8E and 8F). Ectopicexpression of UBE2O in HEK293 cells induced proteasome-mediated MLLdegradation in a dose-dependent manner (FIG. 1E). Knockdown of UBE2O bytwo independent shRNAs had no effect on MLL mRNA expression (FIG. 8G)but increased wild-type MLL protein levels (but not MLL-AF9 proteinlevels) in FLAG-MLL-AF9 HEK293 cells (FIG. 1F). Together, these datademonstrate that UBE2O specifically interacts with the MLL internalregion and induces degradation of wild-type MLL protein.

UBE2O Mediates Interleukin-1 Pathway-Induced MLL Degradation. Toidentify the molecular pathways involved in MLL degradation, we employeda genome-wide shRNA screen. We first generated a stable cell line withrandom integration of Halo-MLL that can fully reconstitute the MLLcomplex (FIGS. 9A and 9B). Cells stained with HaloTag ligand were sortedinto Halo-MLLNeg, Halo-MLLDim, Halo-MLLMid, and Halo-MLLHigh (FIGS. 2Aand 9C). The Halo-MLLDim cells were transduced with the RNAi Consortium(TRC) lentiviral libraries and selected with puromycin for 1-2 weeksbefore performing flow cytometry to sort cells with increased Halo-MLLprotein levels (FIG. 9D). We reproducibly (four of four times) sorted apopulation of cells with elevated Halo-MLL signal in the shRNAlibrary-transduced cells compared to negative control (shGFP-)transducedcells (FIG. 2B). Sequencing of the shRNA sequences from these sortedcells identified 303 gene targets (enriched in at least two differentsortings).

Protein analysis through evolutionary relationships (PANTHER) pathwayanalysis (Mi et al., 2013) of these targets showed that the IL-1 andcytokine receptor activity terms were significantly enriched (FIG. 9E).In the shRNA screening, IL1R1, IL1RAP, and TOLLIP were enriched targets(FIG. 2C). To determine if the IL-1 pathway regulates endogenous MLLprotein, we depleted TOLLIP, MYD88, and IL1RAP within the IL-1 pathway(FIG. 2C shown by stars), and we observed increased levels of endogenousMLL protein (FIGS. 2E and 2F), with no obvious effect on MLLmRNAexpression (FIG. 9F).

We further stimulated HEK293C6 cells, which ectopically express IL1R1and IL1RAP (Lu et al., 2009), with IL-1b in the presence of the proteinsynthesis inhibitor cycloheximide. We found that IL-1b rapidly inducesMLL degradation and increases the MLL-Inter-UBE2O interaction (FIGS. 2Gand 2H). Depletion of UBE2O diminishes IL-1-induced endogenous MLLprotein degradation (FIG. 21), while ectopic expression of UBE2Oincreases MLL-Inter ubiquitination, which could be further elevated byIL-1b stimulation (FIG. 2J). Furthermore, IRAK4 could directlyphosphorylate UBE2O in the in vitro kinase assay (FIG. 2K), raising thepossibility that phosphorylation of UBE2O by IRAK4 could be a regulatorysignal for the enhanced MLLUBE2O interaction and subsequent MLLdegradation induced by IL-1b.

IRAK Inhibition Increases the Stability and Chromatin Occupancy ofWild-Type MLL. Since depleting IRAK4 protein levels can increase MLLprotein levels (FIG. 3A), we asked whether IRAK4's kinase activity wasrequired for MLL degradation. Treating cells with a small moleculeinhibitor of IRAK1/4 (IRAK1/4 inhibitor I), which has been developed toinhibit the IRAK1 and IRAK4 kinase activities (Powers et al., 2006), ledto increased levels of MLL protein in a time- and dose-dependent manner(FIGS. 3B and 10A). A cycloheximide chase assay also demonstrated thatthe IRAK1/4 inhibitor increased MLL protein stability (FIG. 10B).Consistent with the role of the IL-1 pathway and UBE2O in MLLdegradation, IRAK inhibition did not affect MLL-AF9 and MLL-AFF1 proteinlevels (FIGS. 3C and 10C), suggesting that the IRAK activityspecifically signals for the wild-type MLL degradation, but not for theMLL chimeras. Furthermore, we measured the MLL-UBE2O interaction with orwithout IRAK1/4 inhibitor treatment using MudPIT analysis, and we foundthat IRAK inhibition substantially decreased the MLL-UBE2O interaction(FIG. 3D), which was confirmed by co-immunoprecipitation (FIG. 3E).

To investigate the consequences of MLL stabilization and its associationon chromatin, we performed chromatin immunoprecipitation sequencing(ChIP-seq) of MLL in HEK293 cells in the presence or absence of theIRAK1/4 inhibitor. IRAK1/4 inhibitor enhanced MLL occupancy at thewell-characterized MLL target genes, HOXA, HOXC, and FOXC1, as revealedby two different MLL N320 antibodies (CST D2M7U and Bethyl NT86) (FIGS.3F and 10D). Genome-wide analysis demonstrated that IRAK1/4 inhibitionresulted in significant increases in MLL occupancy (FIGS. 3G, 3H, and10D-10F), demonstrating that the MLL protein stabilized by IRAKinhibition can access chromatin.

Stabilization of MLL through IRAK Inhibition and UBE2O DepletionRegulates a Specific Gene Regulatory Network in MLL Leukemia. To measurethe consequence of stabilizing MLL in MLL leukemia cells, we determinedthe effects of IRAK inhibition on cell proliferation of REH(MLL-germline leukemia) and SEM (MLL-AFF1) cells. Both of these celllines are derived from precursor B cell ALL patient blast cells, and,therefore, they have been studied when comparing non-MLL (REH) andMLL-dependent (SEM) leukemia gene expression (Guenther et al., 2008).Treatment with 5 mM IRAK1/4 inhibitor resulted in decreasedproliferation of SEM cells, but not REH cells (FIG. 4A). To avoidpotential off-target effects of the IRAK1/4 inhibitor, we also used asecond inhibitor named IRAK4 inhibitor compound 26, which wascharacterized as a more specific inhibitor of IRAK4 (Tumey et al.,2014). Treatment with 500 nM of this IRAK4 inhibitor led to an evengreater inhibition of SEM cell proliferation and decreased cellviability, with no detectable effect on the REH cells (FIG. 4B).

Total RNA-seq of SEM and REH cells after 2 days of IRAK1/4 or IRAK4inhibitor treatment was performed to characterize gene expressionprofile changes. We found that expression of the IKK/nuclear factor kB(NF-kB) downstream targets HOXA9 and MEIS1 (Kuo et al., 2013) was notreduced by IRAK inhibition in SEM cells, suggesting that IKK/NF-kBsignaling may not be the major target of IRAK inhibition in MLL leukemiacells. However, we found that, in the presence of the IRAK1/4 inhibitor,238 genes were downregulated and 186 genes were upregulated in both REHand SEM cells (FIG. 11A), with the gene ontology analysis of these genesconsistent with a previous report in myelodysplastic syndrome (MDS)cells (FIG. 11B) (Rhyasen et al., 2013). However, this set ofderegulated genes would not explain the different response to IRAKinhibition by the MLL leukemic SEM cells.

To determine the set of genes specifically deregulated in SEM cells, wecompared gene expression changes in SEM and REH cells with both IRAKinhibitors (FIG. 4C). We found 227 downregulated and 119 upregulatedgenes in SEM cells, but not REH cells (FIGS. 4D, 4E, and 11C). Geneontology analysis (Tripathi et al., 2015) of the SEM-specificdownregulated genes showed that cell activation, cellular response togrowth factor stimulus, positive regulation of cell proliferation, andintegrin-mediated signaling pathway were among the top enriched terms(FIGS. 4F and 11D), while no significantly enriched terms were reportedfor the SEM-specific upregulated genes.

Similar to IRAK inhibition, depletion of UBE2O led to a greater defectin SEM cell proliferation compared to REH cells (FIGS. 11E and 11F).Furthermore, ectopic expression of the MLL N terminus (1-1,250 aa),which cannot interact with UBE2O but possesses chromatin-binding domains(FIG. 11G), results in a substantial reduction of SEM cellproliferation, indicating that destabilization of wild-type MLL isrequired for MLL leukemia cell proliferation (FIG. 11H). Our RNA-seqanalysis of SEM cells after UBE2O knockdown found that 121 of the 227genes that were downregulated by the IRAK inhibitors also were decreasedby UBE2O depletion (FIG. 4G). These genes included genes related to cellactivation (LGALS1, GNA15, ALDOA, and EGR1) and cellular response togrowth factor stimulus (P2RY11, SREBF1, RAB13, RAB17, and RAB34) (FIG.4G). Together, these results demonstrate that targeting MLL degradationthrough either IRAK inhibition or UBE2O depletion decreases cellproliferation and downregulates a specific gene regulatory network inMLL leukemia cells.

Determinants of the Increased Sensitivity of MLL Leukemia Cells to IRAKInhibition. To further determine the effectiveness of IRAK inhibitionfor MLL leukemia cell growth, we performed dose-dependent studies withmultiple patient-derived leukemia cell lines, including MLL leukemia andnon-MLL leukemia or lymphoma cells. We found that IRAK4 inhibitortreatment preferentially impeded cell growth of MLL-rearranged AML andALL leukemia cells (FIG. 5A), including the AML MM6 cells for which wedemonstrated similar mRNA expression from the wild-type and translocatedalleles (FIGS. 1A, 1B, and 5B). Interestingly, the MLL-AF6-positive ML2leukemia cells, which have a deletion of the wild-type MLL allele, werenot sensitive to the IRAK4 inhibitor (FIG. 5A). Consistent with thelower sensitivity of ML2 leukemia cells, we found that depletingwild-type MLL in MM6 cells with a shRNA targeting the MLL C terminusreduces the sensitivity of MM6 cells to IRAK inhibition (FIG. 12A).These results suggest that wild-type MLL is required for thepreferential sensitivity of MLL leukemia cells to IRAK inhibition.

Comparing the differentially expressed genes in SEM and MM6 cells afterIRAK inhibition, we found 59 downregulated genes and 28 upregulatedgenes that were shared between both cell lines (FIGS. 5C and 12B). Thesecommon downregulated genes contained genes related to cell activationand cellular response to growth factor stimulus (FIG. 5D). Among thesegenes, LGALS1 and LMO2 previously were identified to be highly expressedin MLL leukemia (Armstrong et al., 2002). Depletion of LGALS1 or LMO2also reduced MM6 cell proliferation (FIGS. 5E and 5F), indicating thatIRAK inhibition could prevent MLL leukemia cell proliferation, at leastpartially, through downregulation of LGALS1 and LMO2 expression.

Using the University of California, Santa Cruz (UCSC) cancer genomedatabase (children's oncology group [COG], POG 9906), we found that IL-1pathway components MYD88, IRAK1, and IRAK4 are expressed at a higherlevel in MLL-rearranged ALL patients than non-MLL ALL patients (FIG.12C). Furthermore, enhanced expression of Irak1 and Irak4was found inprimary and secondary leukemia inanMLL-AF9mousemodel (FIG. 12D) (Liu etal., 2014). Together, these findings indicate a dependence of MLLleukemia on IL-1 signaling, and they suggest that targeting wild-typeMLL degradation by IRAK inhibition is a potential therapeutic approachfor the MLL translocation-based leukemia.

IRAK Inhibition Displaces the MLL Chimera and Subunits of SEC at aSubset of Target Genes. As described above, IRAK inhibitionsignificantly increases MLL chromatin occupancy and preferentiallyimpedes MLL leukemia cell proliferation. We sought to profile thechromatin occupancy of the MLL chimera to determine the mechanisticbasis of IRAK inhibition abrogating the oncogenic potential of MLLchimeras. However, our MLL antibodies recognize the MLL N-terminalepitopes shared by the wild-type MLL and the MLL chimeras. Therefore, weemployed a strategy based on comparing co-occupancy of the MLL fusionpartner AFF1 (AFF1 C-terminal) and the SEC subunits that are recruitedby the MLL chimeras (Lin et al., 2010; Yokoyama et al., 2010). We testedthis strategy in FLAG-MLL-AFF1 HEK293 cells, and we observed increasedoccupancy of MLL-NT at the sites of normal MLL occupancy in HEK293 cells(FIGS. 13A and 13B). The increase of MLLAFF1 chimera occupancy also wasdetected by the co-enrichment of AFF1 (AFF1-CT antibody), which isgenerally not enriched at most of these genes in HEK293 cells (FIGS. 13Aand 13B). Furthermore, SEC subunit recruitment to these

MLL-AFF1-binding sites was demonstrated by AFF4 ChIP-seq (FIGS. 13A and13B). MLL-AFF1 (AFF1-CT) and AFF4 ChIP-seq in SEM cells demonstratedthat MLL-AFF1 and AFF4 occupancies were both reduced in thepromoter-proximal regions of LGALS1, GNA15, and LMO2 genes after IRAKinhibitor treatment (FIG. 6A), consistent with the downregulation ofthese genes by IRAK inhibition. Genome-wide analysis further revealedthat MLL-AFF1 occupancy was significantly decreased (more than 25%)around the promoter-proximal regions of 1,311 genes (FIGS. 6B and 6C),while AFF4 occupancy was decreased at these regions as well (FIGS. 6Dand 6E), demonstrating that IRAK inhibition displaces MLL chimeras andSEC from these chromatin regions.

To further determine if LGALS1, LMO2, and GNA15 are direct target genesof MLL chimeras and SEC, we performed AFF4 knockdown in SEM cells (FIG.13C), and we found that AFF4 depletion resulted in decreased mRNAexpression of LGALS1, LMO2, and GNA15 genes as well (FIGS. 6F and 13D).These results demonstrate that IRAK inhibition displaces MLL chimerasand its oncogenic cofactor SEC at a subset of MLL chimera target genes.

IRAK Inhibitors Substantially Delay the Progression and Improve theSurvival of Murine MLL-AF9 Leukemia In Vivo. We further assessed theeffects of the IRAK inhibitors in vivo using the murine MLL-AF9 leukemiatransplantation model (FIG. 7A) (Volk et al., 2014). Primary MLL-AF9leukemia cells were sensitive to IRAK inhibition based on the decreasedcolony formation and cell proliferation in vitro (FIGS. 14A and 14B). Tomeasure the potential of IRAK inhibitors as a first-line treatment, weinitiated the injection of the animals with IRAK inhibitors on day 19after transplantation, just before they succumb to leukemia.Intraperitoneal injection with IRAK1/4 inhibitor (8 mg/kg), IRAK4inhibitor (75 mg/kg) (Tumey et al., 2014), or vehicle was performedevery other day for 10 days. At sacrifice, the leukemia was confirmed byenlarged spleen, liver weights, elevated white blood cell counts (FIGS.14C-14E), and histological analysis. Both IRAK inhibitors significantlyextended survival of the recipients beyond the 27 days when all of thevehicle-treated mice succumbed to the disease, and they extended thelife of the AML mice to more than 55 days, with one mouse from each IRAKinhibitor-treated group still alive at day 55 (FIG. 7B).

We also treated cohorts of animals 10 days after transplantation (FIG.7C). Strikingly, eight of ten mice from the IRAK1/4 inhibitor-treatedgroup and four of nine mice from the IRAK4 inhibitor group still did notdevelop MLL-AF9 leukemia as of day 55, while all of the vehicle-treatedmice succumbed to the disease by 31 days after transplantation (FIG.7C). IRAK inhibitor treatment of mice led to a substantial decrease ofleukemic blasts in the peripheral blood, as seen by white blood cellcounting (FIG. 14E) or visually in blood smears from the MLL-AF9leukemic mice at the endpoint (FIG. 7D). Wright-Giemsa staining alsorevealed that the blasts from IRAK inhibitor-treated MLL-AF9 leukemicmice were partially differentiated (FIG. 7D). Together, these datasuggest that pharmacologic inhibition of IRAK can delay the progressionand improves survival of the aggressive MLL-AF9 leukemia in vivo.

Discussion

Murine MLL chimera knock-in mice develop leukemia with a long latency,which indicates that multiple cooperating events and/or signalingpathways are required in the pathogenic process (Li and Ernst, 2014).Despite the complexity of these diseases and the remaining challengesfor effective treatments, biochemical and developmental insights haveled to the proposal of several therapeutic strategies, including the useof small molecules that block the Menin-MLL interaction (Borkin et al.,2015) or disrupt the chromatin binding of the bromodomain-containingprotein 4 (BRD4) (Dawson et al., 2011). Inhibitors of themethyltransferase activity of DOT1L also have been tested in phase 1clinical trials, and they are being explored for use in combination withother therapies (Singer, 2015). Recent studies (Fong et al., 2015;Rathert et al., 2015) also reported recurring development of resistanceto BRD4 inhibitors.

Here, we provide a unique mechanism for the treatment of MLLtranslocation-based leukemia via the stabilization of the wild-type copyof MLL (FIG. 7E). Through our biochemical and molecular screens, wedemonstrated that the IL-1 pathway initiates the specific degradation ofwild-type IRAK1/4 and UBE20, while MLL chimeras escape UBE20-mediateddegradation for lack of the MLL-UBE20-interacting region (FIG. 7E). Thehigher expression of IL-1 pathway components in MLL-translocated ALLleukemia patient cells and the increased Irak1 and Irak4 expression inmouse models correlate with the low abundance of wild-type MLL proteinin MLL leukemia cells (FIGS. 1A, 1B, and 8C). The IRAK1/4 inhibitorrecently has been shown to sensitize a subset of MDS and T-ALL cellsthat exhibit high expression of IRAK1 to BCL2 inhibitor treatment,although IRAK1/4 inhibitor alone does not substantially impair thesecells (Li et al., 2015; Rhyasen et al., 2013). We found profound effectsof IRAK inhibition alone for MLL leukemia both in vitro and in vivo.Mechanistically, we found that targeting wild-type MLL degradationthrough IRAK inhibition or UBE2O depletion impedes MLL leukemia cellproliferation and downregulates a common subset of MLL chimera targetgenes. These genes (FIG. 4F) are likely to contribute to the observedantiproliferative effects in MLL leukemia cells. Indeed, knockdown ofLGALS1 or LMO2 leads to decreased MLL leukemia cell proliferation (FIGS.5E and 5F), indicating that downregulation of these genes contributes tothe cell growth inhibition observed after IRAK inhibition or UBE2Oknockdown. The downregulation of these genes could be explained by thedecreased occupancy of the MLL chimeras and the associated SEC, a keycofactor for MLL leukemia (Smith et al., 2011). Together, these findingsexplain the enhanced effects of IRAK inhibition on MLL chimera-drivenleukemia compared to other non-MLL-rearranged leukemia, and they suggestthat stabilization of wildtype MLL could be a potential therapeuticstrategy for MLL leukemia treatment (FIG. 7E).

Although a requirement for wild-type MLL in leukemogenesis has beensuggested by the decreased growth of MLL leukemia cells in the presenceof a small molecule that disrupts the WDRS-MLL SET domain interaction(Cao et al., 2014), a recent study demonstrated that deletion of thewhole SET domain of wild-type MLL has no effect on MLL leukemogenesis,suggesting that at least the HMT activity of MLL is dispensable for MLLleukemogenesis (Mishra et al., 2014). Furthermore, ChIP-seq analysis ofMLL chimera occupancy in ML2 cells, in which the entire wild-type locusis lost, demonstrated that MLL chimeras can access chromatin to mediatetheir oncogenic functions in the absence of wild-type MLL (Okuda et al.,2014; Wang et al., 2011). MLL fusion proteins require an open chromatinstatus for chromatin occupancy due to their impaired chromatin-bindingcapability, likely due to their missing key chromatin-binding modules,such as PHD fingers and a bromodomain (Milne et al., 2010). These datasuggest that, rather than being required for the oncogenic function ofMLL chimeras, wild-type MLL has the potential to outcompete the chimerasthrough additional chromatin-binding modules. Therefore, lessening theimbalance between wild-type MLL and the more abundant oncogenic MLLchimeras could deregulate MLL chimera target gene expression and impairMLL leukemia cell proliferation.

In addition to IL-1 receptors, Toll-like receptors can activate theIRAKs, suggesting that these pathways also may have the potential toregulate MLL stability and contribute to MLL leukemia. In this study, weobserved a few-fold increase of MLL occupancy after IRAK inhibition,with the MLL chimera and SEC occupancy being reduced only at a subset ofgenes generally associated with weak MLL chimera and SEC occupancy.Therefore, searching for additional pathways involved in regulating MLLstability could be very helpful for MLL leukemia treatment. Inconclusion, our study suggests that altering the balance betweenwild-type MLL and MLL oncogenic fusion proteins by modulating signalingpathways is a promising approach for treating the aggressive andrefractory MLL-rearranged leukemia. Furthermore, in addition tostabilization of MLL as a paradigm in the development of therapies foraggressive MLL leukemia, perhaps other cancers caused by translocationscan be treated via developing similar stabilization strategies.

Experimental Models and Subject Details

Cell Lines. HEK293 cells, Flag-MLL-AFF1 and Flag-MLL-AF9 stable celllines (Lin et al., 2010) were cultured in Dulbecco's Modified EagleMedium (DMEM) supplemented with 10% fetal bovine serum (FBS, catalog No.F6178, Sigma). The 293C6 stable cell line with the overexpression ofIL1R1 and IL1RAP in HEK293 cells was a gift from George Stark's Lab(Cleveland Clinic) and maintained in DMEM medium with5% FBS. MONO-MAC-6(MM6, ACC-252), RL (CRL-2261), U-937 (CRL-1593.2), RS4; 11 (CRL-1873),SU-DHL-5 (CRL-2958), SU-DHL-6 (CRL-2959), MOLM13 (ACC-554), OCI-LY1(ACC-722), THP-1 (TIB-202), ML-2 (ACC-15), NB-4 (ACC-207) and REH(ACC-22) leukemia cells were maintained in RPMI-1640 medium supplementedwith 10% FBS. MV4-11 (CRL-9591) and SEM (ACC-546) leukemia cells weremaintained in Iscove's Modified Dulbecco's Medium (IMDM) with 10% FBS.The Mll wildtype Mll+/+ and knockout Mll−/− mouse embryonic fibroblasts(MEF) cell lines were provided by Dr. Jay Hess (University of MichiganMedical School) and cultured in DMEM with 10% FBS.

Expression Plasmids and shRNAs. Mammalian COMPASS expression constructsfused with an N-terminal HaloTag (pFENHK-Halo-MLL, pFENHK-Halo-MLL2(KMT2B), pFENHK-Halo-SETD1A and pFENHK-Halo-MLL4 (KMT2D) plasmids) wereobtained from Promega. Halo-MLL truncates were amplified by PCR withpFENHK-Halo-MLL plasmid and subcloned into the pFENHK plasmid.pCDH-CMV-MLL(1-1250)-EF1-GFP was constructed by insertion of MLL cDNA(1-1250aa) into pCDH-CMV-MCS-EF1-GreenPuro vector (SBI, Cat#: D513B-1).Myc-tagged UBE2O full-length plasmid was provided by El Bachir Affar(University of Montreal). pcDNA5-Flag-UBE2O truncate (552-1292aa) wascloned from UBE2O cDNA (CCSB Human ORFeome UBE2O Clone Without StopCodon Accession: BC051868) (Dharmacon Inc., Clone ID: 11793) into thepcDNA5-Flag plasmid. shRNAs for human TOLLIP (TRCN0000063693 andTRCN0000356024), IL1RAP (TRCN0000058540 and TRCN0000372626), IL1R1(TRCN0000059260 and TRCN0000360115, MYD88 (TRCN0000008025 andTRCN0000011223) were used to knockdown the IL-1 pathway components.IRAK4 was depleted with the shRNAs: TRCN0000002064 and TRCN0000435677.LGALS1 (TRCN0000057423 and TRCN0000057424) and LMO2 (TRCN0000017128 andTRCN0000017130) were depleted with shRNAs in MM6 cells. UBE2O wasdepleted with shRNAs: TRCN0000004587 and TRCN0000272907. Wild-type MLLwas depleted with a shRNA targeting a C-terminal region of MLL(GCCAAGCACTGTCGAAATTAC).

Murine MLL-AF9 Leukemia Model. To generate the MLL-AF9 leukemic mice,bone marrow transplantation with MigR1-MLL-AF9 was performed aspreviously described (Volk et al., 2014). C-Kit+ HSPCs isolated from thebone marrow of female C57BL/6 mice (8-10 weeks old) were spinoculatedwith MigR1-MLL-AF9 and selected with G418 for one week. Primaryrecipient female C57BL/6 mice (Age range from 8-10 weeks) wereirradiated (900 cGy) and transplanted by tail vein intravenous injectionwith 1×106 MLL-AF9 transduced cells along with 2×105 wild-type supportcells. The mice were monitored for signs of acute leukemia for 2-3months and euthanized when leukemia was evident (weight loss below 16.75g, reduced mobility, malaise, palpable spleen, and hunched back).Spleens were isolated from leukemic mice and the homogenate was grown insuspension culture (RPMI-1640 supplemented with penicillin/streptomycin,10% FBS, 100 ng/mL SCF, 50 ng/mL IL6, and 20 ng/mL IL3) for one week.1×104 of the resulting leukemia cells were transplanted intosub-lethally irradiated (450 cGy) female C57BL/6 mice (8-10 weeks) viatail vein injection. Animal studies were approved by the LoyolaUniversity Chicago and Northwestern University Institutional Animal Careand Use Committees.

Method Details

HaloTag Purification and MudPIT Analysis. Plasmids encoding Halo-taggedproteins were transiently transfected into HEK293 cells. 2 days later,HEK293 cells were harvested and lysed with mammalian lysis buffer(Promega). For IRAK1/4 inhibitor treatment, 10 mM IRAK1/4 inhibitor wasadded 24 hr before harvest. Halo-tagged proteins were purified withHaloLink resin in the presence of Benzonase (Sigma) and eluted with TEVprotease. The eluates were precipitated with TCA. After washing withacetone, the protein mixtures were digested with endoproteinase Lys-Cand trypsin (Roche) and analyzed by MudPIT as previously described(Liang et al., 2015a). Original mass spectrometry data can be accessedfrom the Stowers Original Data Repository athttp://www.stowers.org/research/publications/libpb-1090.

Generation of Halo-MLLDim HEK293 Cells. To generate Halo-tagged MLLDimcells, HEK293 cells were transfected with pFENHK-MLL plasmid withPolyethylenimine. 2 days later, the transfected cells were selected with400 ng/ml G418 for 3 weeks and stained with HaloTag R110Direct ligand(Promega). Cells were then sorted according to the Halo-tag signals witha MoFlo sorter (Beckman Coulter) as MLLNegative, MLLDim, MLLMid, andMLLHigh. The sorted cells were grown in DMEM with 10% FBS and selectedwith 400 ng/ml G418. 2 weeks later, the cells were resorted according toHaloTag signals. The MLLDim were further sorted two more times.

IRAK4 Kinase Assay. HEK293 cells were transient transfected with eithervector or pcDNA5-Flag-UBE2O (552-1292aa). 2 days later, Flag-UBE2O waspurified from these HEK293 cells with ANTI-FLAG M2 affinity gel andeluted with FLAG peptides. Eluates from Flag-UBE2O and vector controlpurifications were heat-inactivated and used as substrates inradioactive kinase assays. The phosphorylation of UBE2O were measured inthe presence of 100 ng His-tagged human IRAK4 (Sino Biological), 2 mCig-32P ATP in 20 ml kinase buffer (20 mM HEPES [pH 7.9], 8 mM MgCl2, 0.5%glycerol, 0.1% Triton X-100, 1 mM DTT). After incubation at 37_C for 6hr, reactions were stopped by adding 5 3 SDS loading buffer, and thephosphorylated proteins were visualized by SDS-PAGE and autoradiography.

Genome-wide shRNA Library Screening. A pooled TRC lentiviral library (Miet al., 2013), which contains TRC1 81041 shRNAs and TRC1.5 17563 shRNAs,was used for lentiviral packaging with packaging vectors pD8.9 andpCMV-VSV-G. Lentiviral particles were harvested after 4 days and used totransduce MLLDim HEK293 cells. The lentiviral particles were titered totransduce 30%-50% of the MLLDim cells. The infected cells were furtherselected with 2.0 ng/ml puromycin for 1-2 weeks and stained with HaloTagR110 direct ligand (Promega). Flow cytometry sorting with MoFlo wasperformed to sort for cells with increased Halo-MLL protein levels(Based on the HaloTag signal intensity).

To deconvolve the shRNA composition, the sorted cells were lysed withDirectPCR Lysis Reagent (Viagen Biotech) and shRNA sequences were PCRamplified as a single mixture using vector-backbone directed universalprimers from extracted genomic DNA. 4 independent cell sortings wereperformed representing 4 different biological replicates. The first 3cell sortings were also amplified with a different set of primers torepresent technical replicates. PCR products were cleaned up withQIAGEN's PCR purification kit and digested with Xho I. The 103 bpfragments that contain half-hairpin sequences of the shRNAs were gelpurified and ligated with barcoded linkers. Illumina adapters were addedto PCR products to generate libraries for next-generation sequencing.

The sequence reads were aligned to the TRC reference shRNA library usingBowtie (Langmead et al., 2009) and allowing for 2 mismatches (bowtie-p2-f-v 2-best-strata-m 1). Only uniquely aligned reads were counted andambiguous reads were excluded. The abundance of each shRNA in the sortedcells was based on the number of aligned reads. A shRNA was a “hit” in asort if its abundance was ranked in the top 2× sorted cell number (e.g.,if 400 cells were sorted, a shRNA needed to be among the 800 mostabundant shRNAs). Since each gene in the shRNA library has multipleindependent shRNAs, different shRNAs targeting the same gene wereconsidered in calculating the overall enrichment of a gene. Enrichedgenes had shRNA hits in at least 3 of the 7 sorts to ensure that a hitoccurred in at least two biological replicates.

Chromatin Immunoprecipitation Sequencing. 53107 cells were used per ChIPassay according to a published protocol (Liang et al., 2015b). Briefly,cells were crosslinked with 1% paraformaldehyde for 15 min and werequenched with glycine for 5 min at room temperature. Fixed chromatin wassonicated with a Covaris Focused-ultrasonicator and immune-precipitatedwith the indicated antibody. Libraries were prepared with thehighthrough-put Library preparation kit standard PCR amp module (KAPABiosystems) for next-generation sequencing. ChIP-seq reads were alignedto the mouse (UCSC mm9) or human genome (UCSC hg19). Alignments wereprocessed with Bowtie version 1.1.2, allowing only uniquely mappingreads with up to two mismatches within the 50 bp read. The resultingreads were extended to 150 bases toward the interior of the sequencedfragment and normalized to total reads aligned (reads per million, rpm).For MLL ChIP-seq in 293 cells in FIGS. 3G, 3H, 10E, and 10F, peakdetection was performed with MACS (model-based analysis of ChIP-Seq)version 1.4.2 (Zhang et al., 2008) using default parameters. D2M7U andNT86 peaks that overlapped between the IRAK1/4 inhibitor treated andnon-treated cells were combined and collapsed to give 6,250 MLL-occupiedregions. The average coverage (calculated using rpm tracks describedabove) across the entire region is shown in the boxplots where p valueswere calculated with the Wilcoxon signed-rank test. Heatmaps depict log2 fold change of coverage profiles in a 6 kb window around the mergedpeak center in 25 bp binned averages and sorted by total coverage inthis window.

For FIGS. 6B-6E, heatmaps and metagene plots of the genes with decreasedAFF1-CT read coverage around the TSS (±3 kb) after both IRAK inhibitortreatment were plotted with ngs.plot 2.47 and ranked by read intensity.The AFF4 read coverage at these sites was plotted with the same order.In FIGS. 13A and 13B, all genes with MLL occupancy (12,300 genes) wereplotted in the heatmaps ranked by read intensity. The AFF1-CT and AFF4signals were plotted with the same order. Metagene analysis of MLL,AFF1-CT and AFF4 were calculated based on all of the genes.

Total RNA Sequencing. After IRAK inhibitors treatment for 2 days, REH,SEM and MM6 cells were collected and lysed with Trizol reagent. TotalRNA was extracted from Trizol according to the manufacturer'sinstructions. The RNA was treated with DNase I (NEB) and cleaned withthe QIAGEN RNeasy mini kit. 500 ng RNA was used for library preparationwith TruSeq Stranded Total RNA with Ribo-Zero Gold kit (Illumina,RS-123-2201). The sequenced reads were aligned to the human genome (UCSChgl9) with TopHat 2.1.0 using gene annotations from Ensembl 72.Differential gene expression was performed with EdgeR (Empiricalanalysis of digital gene expression data in R) version 3.08 (Robinson etal., 2010). Adjusted p values were computed using the Benjamini-Hochburgmethod. Protein coding genes, long non-coding RNA and pseudogenes withadjusted p values less than 0.01 were used for the downstream analysiswith Metascape. P values for Venn diagrams were performed with thehypergeometric test.

In vitro MLL-AF9 Assays. Primary murine MLL-AF9 cells were seeded inM3434 methylcellulose (StemCell Technologies, Inc.) at 1×103/10 mm perwell with indicated doses of IRAK1/4, IRAK4 inhibitor, or vehicle andincubated at 37 degrees. Colonies were enumerated after 7 days, anddefined clusters with >100 cells were counted as colonies. For theliquid culture assay, primary MLL-AF9 cells were cultured in media(RPMI-1640 supplemented with penicillin/streptomycin, 10% FBS, 100 ng/mLSCF, 50 ng/mL IL6, and 20 ng/mL IL3), and stained with trypan blue. Theviable cells were counted by a Vi-CELL XR cell counter (BeckmanCoulter).

Animal Treatment. 10 or 19 days after transplantation, mice wererandomized and treated with 8 mg/kg IRAK1/4 inhibitor or 75 mg/kg IRAK4inhibitor, or vehicle (10% DMSO and 90% PBS) every other day for 10days. Mice were monitored for leukemia development and leukemia wasverified after the mice were sacrificed upon signs of illness (weightloss below 16.75 g, partial paralysis, malaise, palpable spleen, andhunched back).

Quantification and Statistical Analyses. Data are presented as Mean±SD.The sample sizes (n) indicate the number of replicates or number of micein each experiment and are provided in the corresponding figure legends.The peak or gene size (N) in the heatmaps indicates the number of peaksor genes included. For FIGS. 5E, 5F, 8G, 9F, and S14, One-Way ANOVAtests were performed with Prism 6 (GraphPad Software, La Jolla, Calif.)to determine the statistical significance. P value <0.005 (**) wasconsidered as highly significantly different, p value <0.05 wasconsidered as significantly different, n.s, not significantly different,p R 0.05. For FIGS. 3H, 6C, 6E, 10F, and 12C, the statisticalsignificance was determined by the Wilcoxon signed-rank test using R3.2.1 package with the p values provided in each figure. For FIGS. 7Band 7C, the Kaplan-Meier survival curves were plotted with GraphPadPrism 6 and the p values were calculated using the log rank test. Pvalues for Venn diagrams in FIG. 4C were calculated with thehypergeometric test in R 3.2.1. For the western blot results,representative figures of at least three biological replicates wereshown. After background subtraction, the densitometric analysis of MLLbands were performed with ImageJ and normalized to the loading controlTubulin. Fold changes of MLL N320 protein were calculated based on thenegative controls as indicated in the figure legends.

Data and Software Availability. The accession number for the raw andprocessed NGS data reported in this paper is GEO: GSE89485. Theaccession numbers for the proteomics data reported in this paper areMSV: 000080298 and PXD: 005233.

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Example 2—Compound Synthesis

The following compounds were synthesized as potential inhibitors ofIRAK4.

Reagent and conditions: (a) tBuOH, 75° C., 47%; (b) NaOH, MeOH; (c)HBTU, DIEPA, DMF, 17% in two steps; (d) MsCl, DIEPA, CHCl₃, AcCN; (e)DMF, 26%-56% in two steps; (f) HCl, THF, 91%.

Reagent and conditions: (a) AcCN, MW, 150° C., 25 min; (b) NH₂NH₂, MW,120° C., 20 min, 68% in two steps; (c) DIEPA, DCM, reflux, 26%; (d)Pd/C, NH₂NH₂, MeOH, 87%.

Reagent and conditions: (a) AcCN, MW, 150° C., 25 min; (b) NH₂NH₂, MW,120° C., 20 min, 28% in two steps; (c) DIEPA, DCM, reflux; (d) Pd/C,NH₂NH₂, MeOH, 58% in two steps.

Reagent and conditions: (a) AcCN, MW, 150C, 25 min; (b) NH₂NH₂, MW, 120°C., 20 min, 28% in two steps; (c) DIEPA, DCM, reflux; (d) Pd/C, NH₂NH₂,MeOH, 58% in two steps.

Reagent and conditions: (a) AcCN, MW, 150° C., 25 min; (b) NH₂NH₂, MW,120° C., 20 min, 63% in two steps; (c) DIEPA, DCM, reflux.

Experimental Section:

Ethyl 7-(hydroxymethyl)imidazo[1,2-a]pyridine-3-carboxylate (3). Amixture of (2-aminopyridin-4-yl)methanol (1, 149 mg, 1.20 mmol) andethyl 2-chloro-3-oxopropanoate (2, 144 mg, 0.956 mmol), in t-butanol (3ml), was stirred at 75° C. under N₂ for 4 h. After cooling down to roomtemperature, solvent was evaporated, and the residue was furtherpurified with flash silica gel column, with EA/Hex (gradient up to99:1), to provide the product as white solid (98 mg, 47%). ¹H NMR (500MHz, Chloroform-d) δ 9.04 (dd, J=7.0, 0.9 Hz, 1H), 8.08 (s, 1H), 7.63(t, J=1.4 Hz, 1H), 6.91 (dd, J=7.1, 1.7 Hz, 1H), 4.71 (d, J=1.3 Hz, 2H),4.32 (q, J=7.1 Hz, 2H), 1.34 (t, J=7.2 Hz, 3H); ¹³C NMR (126 MHz,Chloroform-d) δ 160.43, 148.28, 143.42, 140.81, 127.06, 115.67, 113.44,113.34, 62.91, 60.49, 14.40; ESIMS m/z 221.5 [MH]+.

N-(3-Chlorophenyl)-7-(hydroxymethyl)imidazo[1,2-a]pyridine-3-carboxamide(6). A solution of ethyl7-(hydroxymethyl)imidazo[1,2-a]pyridine-3-carboxylate (3, 201 mg, 0.913mmol) in MeOH (5 ml) was stirred at room temperature. 2 M NaOH aqueoussolution (4.5 mL) was added, and the mixture was carefully acidifiedwith 6 M HCl to pH=5. Remove all solvent, residues combined with HBTU(348 mg, 0.918 mmol) and DIPEA (360 mg, 2.79 mmol), dissolved with dryDMF (4 mL), and stirred under N₂ for 15 min. Then, a solution of3-chloroaniline (190 mg, 1.489 mmol) in dry DMF (2 mL) was added. Themixture was stirred at room temperature overnight. The mixture waspoured into water (25 mL), extracted with ethyl acetate (20 mL×3), theorganic layers were combined, washed with water (20 mL) and brine (20mL) sequentially, dried over Na₂SO₄. Remove solvent, residues furtherpurified by flash silica gel column, with MeOH/DCM (gradient up to15:85) to provide the product as white solid (47 mg, 17%). ¹H NMR (500MHz, Methanol-d₄) δ 9.49 (d, J=7.1 Hz, 1H), 8.46 (s, 1H), 7.91 (t, J=2.1Hz, 1H), 7.70 (s, 1H), 7.61 (dd, J=8.3, 2.0 Hz, 1H), 7.34 (t, J=8.1 Hz,1H), 7.14 (ddd, J=8.7, 6.6, 1.8 Hz, 2H), 4.76 (s, 2H); ¹³C NMR (126 MHz,Methanol-d₄) δ 159.18, 143.84, 139.87, 136.63, 133.98, 129.67, 127.52,123.49, 120.08, 118.31, 113.24, 112.25, 62.19. ESIMS m/z 302.1 [MH]+.

N-(3-Chlorophenyl)-7-(morpholinomethyl)imidazo[1,2-a]pyridine-3-carboxamide(9a, NUCC-200515). A solution ofN-(3-chlorophenyl)-7-(hydroxymethyl)imidazo[1,2-a]pyridine-3-carboxamide(6, 30 mg, 0.099 mmol) and N,N-diisopropylethylamine (0.078 ml, 0.449mmol) in CHCl₃ (4 ml) and acetonitrile (0.5 ml) was stirred at 0° C. Asolution of methanesulfonyl chloride (0.040 ml, 0.511 mmol) in CHCl₃ (1mL) was added. The resulting mixture was stirred at 0° C. for 30 min,warmed to room temperature for 45 min. Solvent was removed, residuesdissolved in dry DMF (2 mL), and a solution of morpholine (8a, 0.091 ml,1.04 mmol) in dry DMF (1 mL) was added. The mixture was heated to 60° C.stirred under N₂ for 2 h. The mixture was diluted with water (20 mL),extracted with ethyl acetate (20 mL×3). The organic layers werecombined, washed with water (20 mL) and brine (20 mL) sequentially,dried over Na₂SO₄. Remove solvent, residues further purified by flashsilica gel column, with MeOH/DCM (gradient up to 15%), to afford theproduct as white solid (20.7 mg, 56%). ¹H NMR (500 MHz, Chloroform-d andMethanol-d₄) δ 9.46 (d, J=7.1 Hz, 1H), 8.42 (s, 1H), 7.84 (t, J=2.0 Hz,1H), 7.63 (s, 1H), 7.59-7.52 (m, 1H), 7.29 (t, J=8.1 Hz, 1H), 7.18 (dd,J=7.2, 1.5 Hz, 1H), 7.10 (ddd, J=8.1, 2.1, 0.9 Hz, 1H), 3.74 (t, J=4.6Hz, 4H), 3.63 (s, 2H), 2.52 (dd, J=5.7, 3.6 Hz, 4H); ¹³C NMR (126 MHz,Chloroform-d and Methanol-d₄) δ 159.28, 139.59, 139.48, 136.91, 134.19,129.71, 127.72, 123.82, 120.48, 118.53, 115.76, 115.52, 66.67, 61.84,53.36. ESIMS m/z 371.2 [MH]+.

N-(3-Chlorophenyl)-7-((4-hydroxypiperidin-1-yl)methyl)imidazo[1,2-a]pyridine-3-carboxamide(9b, NUCC-200550). A solution ofN-(3-chlorophenyl)-7-(hydroxymethyl)imidazo[1,2-a]pyridine-3-carboxamide(6, 17 mg, 0.056 mmol) and N,N-diisopropylethylamine (39 mg, 0.302 mmol)in CHCl₃ (2 ml) and acetonitrile (0.25 ml) was stirred at 0° C. Asolution of methanesulfonyl chloride (26 mg, 0.227 mmol) in CHCl₃ (0.5mL) was added. The resulting mixture was stirred at 0° C. for 30 min,warmed to room temperature for 45 min. Solvent was removed, residuescombined with piperidin-4-ol (8b, 51.2 mg, 0.506 mmol), dissolved in dryDMF (2 mL). The mixture was heated to 60° C. stirred under N₂ for 2 h.Remove solvent in vacuo, residues further purified flash silica gelcolumn, with MeOH/DCM (gradient up to 20:80), to afford the product aswhite solid (10.5 mg, 48%). ¹H NMR (500 MHz, Methanol-d4) δ 9.48 (d,J=7.1 Hz, 1H), 8.41 (s, 1H), 7.85 (t, J=2.1 Hz, 1H), 7.66-7.52 (m, 2H),7.31 (t, J=8.1 Hz, 1H), 7.18 (dd, J=7.2, 1.7 Hz, 1H), 7.12 (dd, J=7.9,2.0 Hz, 1H), 3.75-3.52 (m, 2H), 3.39 (m, 1H), 3.06 (dt, J=13.1, 4.2 Hz,2H), 2.83 (dt, J=11.8, 4.8 Hz, 2H), 2.60 (ddd, J=13.3, 10.8, 2.9 Hz,2H), 2.33-2.14 (m, 2H); ¹³C NMR (126 MHz, Chloroform-d and Methanol-d₄)δ 159.34, 147.89, 139.92, 139.52, 136.93, 134.28, 129.75, 127.68,123.94, 120.61, 118.61, 118.42, 115.86, 115.66, 67.28, 61.66, 43.72,34.84. ESIMS m/z 385.5 [MH]+.

tert-Butyl4-((3-((3-chlorophenyl)carbamoyl)imidazo[1,2-a]pyridin-7-yl)methyl)piperazine-1-carboxylate(9c, NUCC-200552. A solution ofN-(3-chlorophenyl)-7-(hydroxymethyl)imidazo[1,2-a]pyridine-3-carboxamide(6, 17 mg, 0.056 mmol) and N,N-diisopropylethylamine (26 mg, 0.227 mmol)in CHCl₃ (0.5 ml) and acetonitrile (0.25 ml) was stirred at 0° C. Asolution of methanesulfonyl chloride (26 mg, 0.227 mmol) in CHCl₃ (0.5mL) was added. The resulting mixture was stirred at 0° C. for 30 min,warmed to room temperature for 45 min. Solvent was removed, residuescombined with tert-butyl piperazine-1-carboxylate (8c, 63.6 mg, 0.341mmol) dissolved in dry DMF (2 mL). The mixture was heated to 60° C.stirred under N₂ for 2 h. Remove solvent in vacuo, residues furtherpurified by preparative HPLC, with AcCN/H₂O, to afford the product aswhite solid (6.9 mg, 26%). ¹H NMR (500 MHz, Chloroform-d nd Methanol-d4)δ 9.49 (d, J=7.1 Hz, 1H), 8.45 (s, 1H), 7.90 (t, J=2.1 Hz, 1H), 7.67 (s,1H), 7.64-7.54 (m, 1H), 7.33 (t, J=8.1 Hz, 1H), 7.23 (dd, J=7.2, 1.8 Hz,1H), 7.18-7.07 (m, 1H), 3.72 (s, 2H), 3.48 (t, J=4.9 Hz, 4H), 2.52 (t,J=5.0 Hz, 4H), 1.45 (s, 9H). ESIMS m/z 470.5 [MH]+.

N-(3-Chlorophenyl)-7-(piperazin-1-ylmethyl)imidazo[1,2-a]pyridine-3-carboxamide(9d, NUCC-200551). A solution of tert-butyl4-((3-((3-chlorophenyl)carbamoyl)imidazo[1,2-a]pyridin-7-yl)methyl)piperazine-1-carboxylate(9c, 5.8 mg, 0.012 mmol) and TFA (100 mg, 0.307 mmol) in DCM (1.5 mL)was stirred at room temperature overnight. Solvent was removed, andresidues was washed with ether to afford product as white yellow solidas TFA salt (6.7 mg, 91%). ¹H NMR (500 MHz, Methanol-d4) δ 9.74 (d,J=7.2 Hz, 1H), 8.70 (s, 1H), 8.04 (s, 1H), 7.95 (t, J=2.1 Hz, 1H),7.71-7.57 (m, 1H), 7.43 (d, J=7.2 Hz, 1H), 7.38 (t, J=8.1 Hz, 1H), 7.18(ddd, J=7.9, 2.1, 0.9 Hz, 1H), 4.51 (s, 2H), 3.73 (s, 4H). ESIMS m/z370.5 [MH]+.

5-(3-Nitrophenyl)-1H-imidazol-2-amine (13). A solution of2-bromo-1-(3-nitrophenyl)ethanone (10, 651 mg, 2.67 mmol) andpyrimidin-2-amine (11, 261 mg, 2.74 mmol) in acetonitrile (3 mL) washeated to 150° C. in a microwave reactor for 25 min. After cooling downto room temperature, hydrazine monohydrate (0.5 mL, 9.66 mmol) was addedand the resulting mixture was heated to 120° C. in a microwave reactorfor 20 min. The solid was removed by filtration, washed withacetonitrile. The filtrate was concentrated and further purified byflash silica gel column, with MeOH/DCM (gradient up to 15:85), to affordthe product as dark brown oil (370 mg, 68%). ¹H NMR (500 MHz,Chloroform-d and Methanol-d4) δ 8.36 (t, J=2.0 Hz, 1H), 8.04-7.90 (m,1H), 7.86 (dt, J=7.9, 1.3 Hz, 1H), 7.45 (t, J=8.0 Hz, 1H), 6.98 (s, 1H);¹³C NMR (126 MHz, Chloroform-d and Methanol-d4) δ 150.65, 148.58,135.47, 129.58, 129.39, 120.20, 118.27. ESIMS m/z 205.1 [MH]+.

3-Chloro-N-(5-(3-nitrophenyl)-1H-imidazol-2-yl)benzamide (15). Asolution of 5-(3-nitrophenyl)-1H-imidazol-2-amine (13, 102 mg, 0.500mmol) and DIEPA (293 mg, 2.267 mmol) in DCM (10 mL) was stirred at roomtemperature, then a solution of 3-chlorobenzoyl chloride (14, 320 mg,1.828 mmol) in DCM (10 mL) was added. The mixture was heated to refluxfor 72 h. The solvent was evaporated, residues further purified by flashsilica gel column, with EA/hex (gradient up to 60:40) to afford theproduct as yellow solid (45 mg, 26%). ¹H NMR (500 MHz, Chloroform-d andMethanol-d4) δ 8.50 (s, 1H), 8.07 (dd, J=8.2, 2.3 Hz, 1H), 8.04-7.95 (m,2H), 7.91-7.84 (m, 1H), 7.60-7.52 (m, 2H), 7.46 (t, J=7.9 Hz, 1H), 7.29(s, 1H). ESIMS m/z 343.4 [MH]+.

N-(5-(3-aminophenyl)-1H-imidazol-2-yl)-3-chlorobenzamide (16,NUCC-200554). A solution of3-chloro-N-(5-(3-nitrophenyl)-1H-imidazol-2-yl)benzamide (15, 6.3 mg,0.018 mmol) and hydrazine monohydrate (57 mg, 1.07 mmol) in methanol wasstirred at room temperature, 10% Pd/C (3.0 mg) was added. The mixturewas heated to 80° C. for 10 min. The solid was removed by filtration.The filtrate was concentrated, and further purified by prep HLPC, withAcCN/H₂O, to afford the product as white solid (5.0 mg, 87%). ¹H NMR(500 MHz, Chloroform-d and Methanol-d4) δ 8.04 (s, 1H), 7.94 (d, J=7.7Hz, 1H), 7.57 (dd, J=8.0, 1.9 Hz, 1H), 7.48 (t, J=7.9 Hz, 1H), 7.20-7.08(m, 2H), 7.03 (t, J=5.7 Hz, 2H), 6.66 (dd, J=7.9, 2.1 Hz, 1H); ¹³C NMR(126 MHz, Chloroform-d and Methanol-d4) δ 147.33, 134.43, 131.81,129.78, 129.34, 127.92, 125.95, 114.76, 114.37, 111.38. ESIMS m/z 313.4[MH]+.

3-(2-Amino-1H-imidazol-4-yl)phenol (19). A solution of2-bromo-1-(3-hydroxyphenyl)ethan-1-one (17, 126 mg, 0.562 mmol) andpyrimidin-2-amine (11, 58 mg, 0.591 mmol) in acetonitrile (1 mL) washeated to 150° C. in a microwave reactor for 25 min. After cooling downto room temperature, hydrazine monohydrate (0.2 mL, 3.8 mmol) was addedand the resulting mixture was heated to 120° C. in a microwave reactorfor 20 min. The mixture was concentrated and further purified bypreparative HPLC, with AcCN/H₂O, to provide the product as pale yellowoil (28 mg, 28%). ¹H NMR (500 MHz, Methanol-d4) δ 7.13 (t, J=7.9 Hz,1H), 7.04 (d, J=7.7 Hz, 1H), 7.01 (t, J=2.0 Hz, 1H), 6.84 (s, 1H), 6.62(dd, J=8.1, 2.4 Hz, 1H); ¹³C NMR (126 MHz, Methanol-d4) δ 157.30,150.32, 134.39, 129.15, 114.97, 112.64, 110.33. ESIMS m/z 176.3 [MH]+.

N-(5-(3-hydroxyphenyl)-1H-imidazol-2-yl)benzamide (21, NUCC-200618). Asolution of 3-(2-amino-1H-imidazol-4-yl)phenol (19, 28 mg, 0.16 mmol)and DIEPA (147 mg, 1.14 mmol) in DCM (10 mL) was stirred at roomtemperature, then a solution of 3-chlorobenzoyl chloride (14, 142 mg,0.787 mmol) in DCM (10 mL) was added. The mixture was heated to refluxovernight. Solvent was evaporated, residues combined with 10% Pd/C (37mg) and hydrazine monohydrate (389 mg, 7.28 mmol), dissolved withmethanol (10 mL). The resulting mixture was heated to 80° C. for 30 min.The solid was remove by filtration, the filtrate was concentrated andpurified by flash silica gel column, with ethylacetate/hexanes (gradientup to 99:1), to afford the product as white solid (26 mg, 58%). ¹H NMR(500 MHz, Chloroform-d and Methanol-d4) δ 7.94-7.87 (m, 2H), 7.52 (dd,J=8.5, 6.5 Hz, 1H), 7.47-7.41 (m, 2H), 7.14 (t, J=7.9 Hz, 1H), 7.07-7.03(m, 1H), 7.02 (d, J=2.8 Hz, 2H), 6.66 (dd, J=8.0, 2.5 Hz, 1H); ¹³C NMR(126 MHz, Chloroform-d and Methanol-d4) δ 167.21, 157.18, 132.87,132.53, 129.81, 128.65, 127.59, 116.01, 114.09, 111.32. ESIMS m/z 280.4[MH]+.

3-(2-Amino-1H-imidazol-4-yl)benzonitrile (24). A solution of3-(2-bromoacetyl)benzonitrile (22, 462 mg, 2.06 mmol) andpyrimidin-2-amine (11, 197 mg, 2.07 mmol) in acetonitrile (2 mL) washeated to 150° C. in a microwave reactor for 25 min. After cooling downto room temperature, hydrazine monohydrate (0.4 mL, 7.73 mmol) was addedand the resulting mixture was heated to 120° C. in a microwave reactorfor 20 min. The solid was removed by filtration, washed withacetonitrile. The filtrate was concentrated and further purified bypreparative HPLC, with AcCN/H₂O, to provide the product as yellow oil(69 mg, 18%). ¹H NMR (500 MHz, Methanol-d4) δ 7.90 (s, 1H), 7.84 (dt,J=4.7, 2.3 Hz, 1H), 7.45 (d, J=5.9 Hz, 2H), 7.06 (s, 1H); ¹³C NMR (126MHz, Methanol-d4) δ 151.03, 135.26, 132.70, 129.22, 128.65, 127.83,126.76, 118.58, 112.13, 111.43. ESIMS m/z 185.2 [MH]+.

3-Chloro-N-(5-(3-cyanophenyl)-1H-imidazol-2-yl)benzamide (25,NUCC-200617). A solution of 3-(2-amino-1H-imidazol-4-yl)benzonitrile(24, 69 mg, 0.38 mmol) and DIEPA (182 mg, 1.41 mmol) in DCM (10 mL) wasstirred at room temperature, then a solution of 3-chlorobenzoyl chloride(14, 175 mg, 0.97 mmol) in DCM (5 mL) was added. The mixture was heatedto reflux overnight. The solvent was evaporated, residues furtherpurified by flash silica gel column, with EA/hex (gradient up to 99:1)to afford the product as yellow solid (23 mg, 19%). ¹H NMR (500 MHz,Chloroform-d and Methanol-d4) δ 7.96-7.88 (m, 2H), 7.85 (d, J=7.7 Hz,1H), 7.81 (dt, J=7.9, 1.3 Hz, 1H), 7.50 (dt, J=8.3, 1.3 Hz, 1H),7.48-7.37 (m, 3H), 7.18 (s, 1H). ESIMS m/z 323.3 [MH]+.

N-(5-(3-carbamoylphenyl)-1H-imidazol-2-yl)-3-chlorobenzamide (26,NUCC-200619). A suspension of3-chloro-N-(5-(3-cyanophenyl)-1H-imidazol-2-yl)benzamide (25, 9.0 mg,0.028 mmol) and potassium carbonate (90 mg, 0.651 mmol) in DMSO wasstirred at room temperature under N₂. Hydrogen peroxide (0.090 ml, 0.882mmol) and magnesium oxide (12 mg, 0.298 mmol) was added sequentially.The resulting mixture was stirred at room temperature for 4 h, andpoured into water (5 mL), and extracted with ethyl acetate (8 mL×5). Theorganic layers were combined, washed with water (10 mL) and brine (10mL) sequentially, dried over Na₂SO₄. Remove solvent, the solid wastriturated with ether (10 mL), to afford the product as pale yellowsolid (5.1 mg, 54%). ESIMS m/z 341.3 [MH]+. ¹H NMR (500 MHz,Methanol-d4) δ 8.54 (s, 1H), 8.23 (s, 1H), 8.15 (d, J=7.8 Hz, 1H), 8.02(s, 1H), 7.90 (d, J=8.0 Hz, 1H), 7.79 (d, J=7.6 Hz, 1H), 7.63 (t, J=7.9Hz, 1H), 7.58 (d, J=7.5 Hz, 1H), 7.47 (t, J=7.9 Hz, 1H).

5-(4-Chlorophenyl)-1H-imidazol-2-amine (29). A solution of2-bromo-1-(4-chlorophenyl)ethanone (27, 526 mg, 2.25 mmol) andpyrimidin-2-amine (11, 225 mg, 2.37 mmol) in acetonitrile (3 mL) washeated to 150° C. in a microwave reactor for 25 min. After cooling downto room temperature, hydrazine monohydrate (0.4 mL, 7.73 mmol) was addedand the resulting mixture was heated to 120° C. in a microwave reactorfor 20 min. The solid was removed by filtration, washed withacetonitrile. The filtrate was concentrated and further purified byflash silica gel column, with MeOH/DCM (gradient up to 15:85), to affordthe product as dark brown oil (273 mg, 63%). ¹H NMR (500 MHz,Methanol-d4) δ 7.64-7.44 (m, 2H), 7.27 (d, J=8.4 Hz, 2H), 6.91 (s, 1H);¹³C NMR (126 MHz, Methanol-d4) δ 151.15, 149.10, 130.73, 129.34, 126.61,123.39. ESIMS m/z 194.0 [MH]+.

3-Chloro-N-(5-(4-chlorophenyl)-1H-imidazol-2-yl)benzamide (31a). Asolution of 5-(4-chlorophenyl)-1H-imidazol-2-amine (29, 34 mg, 0.176mmol) and DIEPA (104 mg, 0.80 mmol) in DCM (4 mL) was stirred at roomtemperature, then a solution of 3-chlorobenzoyl chloride (14, 66 mg,0.37 mmol) in DCM (1 mL) was added. The mixture was heated to refluxovernight. The solvent was evaporated, residues further purified byflash silica gel column, with EA/hex (gradient up to 50:50) to affordthe product as white yellow solid (25 mg, 43%). ¹H NMR (500 MHz,Methanol-d4) δ 7.96 (t, J=1.9 Hz, 1H), 7.84 (dt, J=7.5, 1.5 Hz, 1H),7.58-7.49 (m, 3H), 7.42 (t, J=7.9 Hz, 1H), 7.31 (d, J=8.7 Hz, 2H), 7.08(s, 1H); ¹³C NMR (126 MHz, Chloroform-d and Methanol-d4) δ 161.79,138.64, 130.84, 130.65, 128.62, 128.50, 126.01, 124.83, 123.98, 121.89,121.81. ESIMS m/z 332.0 [MH]+.

N-(5-(4-Chlorophenyl)-1H-imidazol-2-yl)-2-methoxybenzamide (31b). Asolution of 5-(4-chlorophenyl)-1H-imidazol-2-amine (29, 37.3 mg, 0.156mmol) and DIEPA (99 mg, 0.77 mmol) in DCM (6 mL) was stirred at roomtemperature, then a solution of 2-methoxybenzoyl chloride (30, 44 mg,0.26 mmol) in DCM (2 mL) was added. The mixture was heated to refluxovernight. The solvent was evaporated, residues further purified byflash silica gel column, with EA/hex (gradient up to 60:40) to affordthe product as white yellow solid (40.1 mg, 79%). ¹H NMR (500 MHz,Chloroform-d) δ 7.54-7.44 (m, 3H), 7.38 (dd, J=7.5, 1.8 Hz, 1H),7.27-7.17 (m, 2H), 7.05 (td, J=7.5, 0.9 Hz, 1H), 7.00 (d, J=8.4 Hz, 1H),6.58 (s, 1H); ¹³C NMR (126 MHz, Chloroform-d) δ 168.01, 156.34, 150.93,137.29, 133.13, 133.01, 131.27, 128.77, 128.68, 126.33, 123.22, 120.85,111.68, 107.64, 55.84. ESIMS m/z 328.1 [MH]+.

Example 3—Compound Testing

The newly synthesized compounds were tested in an IRAK4 kinaseinhibition assay as follows. In a 10 μl reaction, 10 ng Human IRAK4,12.5 μM ATP, 500 ng dephosphorylated MBP (EMD Millipore), and differentIRAK inhibitors were used in 1× kinase buffer (60 mM HEPES-NaOH, pH 7.5,3 mM MgCl2, 3 mM MnCl2, 1.2 mM DTT, 0.1% BSA) at room temperature for 45minutes. The ADP formed from the kinase reaction was measured by theADP-Glo™ Kinase Assay (Cat.#V9101) and the luminescence was measuredwith a Tecan Infinite M1000 plate reader. As indicated in FIG. 15,Compound NUCC0200554 (Compound 16 of Example 2) and Compound NUCC0200618(Compound 21 of Example 2) were observed to inhibit IRAK4 kinaseactivity.

The newly synthesized compound NUCC0200554 (Compound 16 of Example 2)and compound NUCC0200618 (Compound 21 of Example 2) were further testedin MLL stabilization assay, in an IRAK4 kinase activity as describedabove, and in a SEM cell proliferation assay as follows. SEM (ACC-546)leukemia cells were maintained in Iscove's Modified Dulbecco's Medium(IMDM) with 10% FBS. Viable SEM cells were seeded at 0.2 million/ml, andtreated with different concentrations of inhibitors at the indicatedconcentrations for three days. Viable cells were monitored by trypanblue exclusion staining and counted using a Vi-CELL XR cell counter.Data are represented as Mean±SD (n=3). The results provided in FIG. 16demonstrate that the tested compounds stabilize MLL, inhibit IRAK4kinase, and inhibit SEM cell proliferation at increasing concentration.

The gene expression profiles of SEM cells treated with newly synthesizedcompound NUCC0200554 (Compound 16 of Example 2) and compound NUCC0200618(Compound 21 of Example 2) as follows. After inhibitor treatment for 3days, SEM cells were collected and lysed with Trizol reagent. Total RNAwas extracted from Trizol according to the manufacturer's instructions.The RNA was treated with DNase I (NEB) and cleaned with the QiagenRNeasy mini kit. 500 ng RNA was used for library preparation with TruSeqStranded Total RNA with Ribo-Zero Gold kit (Illumina, RS-123-2201). Thesequenced reads were aligned to the human genome (UCSC hgl9) with TopHat2.1.0 using gene annotations from Ensembl 72. Differential geneexpression was performed with Deseq2. The results are provided in FIG.17.

In the foregoing description, it will be readily apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention. The invention illustratively described hereinsuitably may be practiced in the absence of any element or elements,limitation or limitations which is not specifically disclosed herein.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention. Thus, it should be understood that although the presentinvention has been illustrated by specific embodiments and optionalfeatures, modification and/or variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention.

Citations to a number of patent and non-patent references are madeherein. The cited references are incorporated by reference herein intheir entireties. In the event that there is an inconsistency between adefinition of a term in the specification as compared to a definition ofthe term in a cited reference, the term should be interpreted based onthe definition in the specification.

We claim:
 1. A method for treating a cancer characterized by arearrangement in the mixed lineage leukemia gene (MLL-r) in a subject inneed thereof, wherein the cancer is MLL-r leukemia, the methodcomprising administering a therapeutic amount of a therapeutic agentthat inhibits the biological activity of interleukin-1receptor-associated kinase 4 (IRAK4), wherein the therapeutic agentcomprises a compound having a formula:

wherein R², R³, and R⁴ are the same or different and each of R², R³, andR⁴ is independently —(CH₂)_(m)R′, wherein m is 0-6 and R′ is selectedfrom hydrogen, halo, amino or alk-substituted amino, hydroxyl, cyano,nitro, alkyl which may be straight chain or branched, allyl, alkoxywhich may be straight chain or branched, saturated or unsaturatedcycloalkyl which optionally is substituted with alkyl, halo, hydroxyl,or amino, and saturated or unsaturated heterocycloalkyl which optionallyis substituted with alkyl, halo, hydroxyl, or amino; and wherein R⁷, R⁸,and R⁹ are the same or different and each of R⁷, R⁸, and R⁹ isindependently —(CH₂)_(n)R″, wherein n is 0-6 and R″ is selected fromhydrogen, halo, amino or alkyl-substituted amino, hydroxyl, cyano,nitro, alkyl which may be straight chain or branched, allyl, alkoxywhich may be straight chain or branched, saturated or unsaturatedcycloalkyl which optionally is substituted with alkyl, halo, hydroxyl,or amino, and saturated or unsaturated heterocycloalkyl which optionallyis substituted with alkyl, halo, hydroxyl, or amino.
 2. A compoundhaving a formula:

wherein R², R³, and R⁴ are the same or different and each of R², R³, andR⁴ is independently —(CH₂)_(m)R′, wherein m is 0-6 and R′ is selectedfrom hydrogen, halo, amino or alkyl-substituted amino, hydroxyl, cyano,nitro, alkyl which may be straight chain or branched, allyl, alkoxywhich may be straight chain or branched, saturated or unsaturatedcycloalkyl which optionally is substituted with alkyl, halo, hydroxyl,or amino, and saturated or unsaturated heterocycloalkyl which optionallyis substituted with alkyl, halo, hydroxyl, or amino; and wherein R⁷, R⁸,and R⁹ are the same or different and each of R⁷, R⁸, and R⁹ isindependently —(CH₂)_(n)R″, wherein n is 0-6 and R″ is selected fromhydrogen, halo, amino or alkyl-substituted amino, hydroxyl, cyano,nitro, alkyl which may be straight chain or branched, allyl, alkoxywhich may be straight chain or branched, saturated or unsaturatedcycloalkyl which optionally is substituted with alkyl, halo, hydroxyl,or amino, and saturated or unsaturated heterocycloalkyl which optionallyis substituted with alkyl, halo, hydroxyl, or amino.
 3. The compound ofclaim 2, wherein at least one of R², R³, R⁴, R⁷, R⁸, and R⁹ is nothydrogen.
 4. The compound of claim 2, wherein at least one of R², R³,and R⁴ is halo or alkyoxy and at least one of R⁷, R⁸, and R⁹ is amino,alkyl-substituted amino, dialkyl-substituted amino, amido, cyano, nitro,hydroxyl, or halo.
 5. The compound of claim 2, wherein the compound hasa formula selected from:


6. The compound of claim 2, wherein the compound has a formula selectedfrom:


7. A pharmaceutical composition comprising the compound of claim 2 and apharmaceutically acceptable carrier.
 8. A pharmaceutical compositioncomprising the compound of claim 3 and a pharmaceutically acceptablecarrier.
 9. A pharmaceutical composition comprising the compound ofclaim 4 and a pharmaceutically acceptable carrier.
 10. A pharmaceuticalcomposition comprising the compound of claim 5 and a pharmaceuticallyacceptable carrier.
 11. A pharmaceutical composition comprising thecompound of claim 6 and a pharmaceutically acceptable carrier.
 12. Amethod for treating MLL-r leukemia in a subject in need thereof, themethod comprising administering to the subject the pharmaceuticalcomposition of claim
 7. 13. A method for treating MLL-r leukemia in asubject in need thereof, the method comprising administering to thesubject the pharmaceutical composition of claim
 8. 14. A method fortreating MLL-r leukemia in a subject in need thereof, the methodcomprising administering to the subject the pharmaceutical compositionof claim
 9. 15. A method for treating MLL-r leukemia in a subject inneed thereof, the method comprising administering to the subject thepharmaceutical composition of claim
 10. 16. A method for treating MLL-rleukemia in a subject in need thereof, the method comprisingadministering to the subject the pharmaceutical composition of claim 11.